UNIVERSIDADE FEDERAL DE SERGIPE

PRÓ-REITORIA DE PÓS-GRADUAÇÃO E PESQUISA

MESTRADO EM CIÊNCIAS FARMACÊUTICAS

SÍNTESE DO PROPIONATO DE CARVACROL E ESTUDO

DE SUAS PROPRIEDADES ANTI-HIPERALGÉSICA E

ANTI-INFLAMATÓRIA EM PROTOCOLOS

EXPERIMENTAIS

Marilia Trindade de Santana Souza

São Cristóvão-SE

2014

UNIVERSIDADE FEDERAL DE SERGIPE

PRÓ-REITORIA DE PÓS-GRADUAÇÃO E PESQUISA

MESTRADO EM CIÊNCIAS FARMACÊUTICAS

SÍNTESE DO PROPIONATO DE CARVACROL E ESTUDO

DE SUAS PROPRIEDADES ANTI-HIPERALGÉSICA E

ANTI-INFLAMATÓRIA EM PROTOCOLOS

EXPERIMENTAIS

Marilia Trindade de Santana Souza

Dissertação apresentada ao Núcleo de Pós-Graduação em Ciências Farmacêuticas da Universidade Federal de Sergipe como requisito parcial à obtenção do grau de Mestre em Ciências Farmacêuticas.

Orientador: Prof. Dr. Lucindo José Quintans Júnior

Co-orientador: Prof. Dr. Sócrates Cabral de H. Cavalcanti

São Cristóvão-SE

2014

i

SO

UZ

A, M

.T.S

. S

ínte

se d

o p

rop

iona

to d

e c

arv

acro

l e

estu

do d

e s

uas p

roprie

da

de

s a

nti-h

ipera

lgésic

a e

anti-in

flam

ató

ria e

m

pro

tocolo

s e

xpe

rim

enta

is.

201

4.

ii

FICHA CATALOGRÁFICA ELABORADA PELA BIBLIOTECA CENTRAL

UNIVERSIDADE FEDERAL DE SERGIPE

S729s

Souza, Marilia Trindade de Santana Síntese do propionato de carvacrol e estudo de suas

propriedades anti-hiperalgésica e anti-inflamatória em protocolos experimentais / Marilia Trindade de Santana Souza ; orientador Lucindo José Quintas Júnior. – São Cristóvão, 2014.

111 f. : il.

Dissertação (mestrado em Ciências Farmacêuticas)–Universidade Federal de Sergipe, 2014.

1. Carvacrol. 2. Monoterpeno. 3. Agentes anti-inflamatórios. 4. Hiperalgesia. I. Quintas Júnior, Lucindo José, orient. II. Título.

CDU 615.276:582.929.4

iii

Marilia Trindade de Santana Souza

SÍNTESE DO PROPIONATO DE CARVACROL E ESTUDO

DE SUAS PROPRIEDADES ANTI-HIPERALGÉSICA E

ANTI-INFLAMATÓRIA EM PROTOCOLOS

EXPERIMENTAIS

Dissertação apresentada ao Núcleo de Pós-Graduação em Ciências Farmacêuticas da Universidade Federal de Sergipe como requisito parcial à obtenção do grau de Mestre em Ciências Farmacêuticas.

Aprovada em: _____/_____/_____

________________________________________________

Orientador: Professor Dr Lucindo José Quintans Júnior

_________________________________________________

1º Examinador: Dr Jackson Roberto Guedes da Silva Almeida

_________________________________________________

2° Examinador: Dr Cristiani Isabel Banderó Walker

PARECER

-----------------------------------------------------------------------------------------------------

-----------------------------------------------------------------------------------------------------

-----------------------------------------------------------------------------------------------------

iv

DEDICATÓRIA

Dedico este trabalho a Deus, pois sem

Ele eu nada seria. “Minha força e

vitória tem um nome e é Jesus!” (Autor

Desconhecido).

v

AGRADECIMENTOS

A Deus, pela força e sabedoria que me guia e protege.

À minha mãe Maria Celeste Trindade pelo amor incondicional, me ajudando a vencer

todos os obstáculos e a alcançar essa tão sonhada conquista. Amo muito você!

Ao meu esposo Pedro Mendes de Souza, pela paciência e compreensão por todos os

momentos de ausência. Principalmente, por acreditar que sou capaz de realizar as coisas

quando nem eu mesmo acredito. Obrigada, Amo você!

À Universidade Federal de Sergipe e todo o seu corpo docente, por formarem o

profissional que sou hoje.

Ao Prof. Dr. Lucindo José Quintans Júnior, meu agradecimento pela oportunidade da

realização deste trabalho, sobretudo, agradeço pelo apoio, paciência e pelo exemplo de

determinação, competência e dedicação.

Ao Prof Dr. Sócrates Cabral de Holanda Cavalcanti, pela ajuda na realização deste

trabalho, pela paciência, incentivo, e por ter me proporcionado um grande crescimento

científico, principalmente na área de Química Farmacêutica.

Ao Prof. Dr. Enilton Camargo, pelos ensinamentos que contribuíram com a minha

formação, sendo um modelo de competência e dedicação.

Ao Prof. Dr. Emiliano Oliveira Barreto, da Universidade Federal de Alagoas, por

possibilitar a realização de parte desse trabalho.

vi

A todos os integrantes do Laboratório de Farmacologia Pré-clínica (LAPEC).

Aos meus amigos, Douglas Prado, Makson Oliveira, Mônica Santos Melo, Priscila Laise,

Renan Guedes pela amizade, carinho, colaboração e evolução conjunta, tornando cada

experimento finalizado uma vitória. Obrigada pelo apoio nos momentos difíceis, sempre

me fortalecendo a cada obstáculo.

Às grandes amigas de curso Daniele, Gabriela, Magda, Viviane pela amizade,

compreensão, apoio e cumplicidade, levarei vocês sempre no meu coração.

Ao Sr. José Osvaldo Andrade Santos pelo suporte técnico realizado no Biotério.

Ao CNPq pela bolsa de estudo concedida, à FAPITEC e à CAPES pelo auxílio financeiro.

Muito Obrigada!

Marilia Trindade de Santana Souza

vii

RESUMO

SANTANA, M.T. SÍNTESE DO PROPIONATO DE CARVACROL E ESTUDO DE

SUAS PROPRIEDADES ANTI-HIPERALGÉSICA E ANTI-INFLAMATÓRIA EM

PROTOCOLOS EXPERIMENTAIS Dissertação de Mestrado em Ciências

Farmacêuticas, Universidade Federal de Sergipe, 2014.

Os terpenos são compostos naturais obtidos do metabolismo secundário das plantas.

Apesar de apresentar efeitos farmacológicos, modificações estruturais realizadas no seu

esqueleto podem levar o aumentando de suas atividades farmacológicas e atenuar os

efeitos toxicológicos. Neste contexto, insere-se o carvacrol, um monoterpeno fenólico,

presente em óleos essenciais de plantas pertencentes à família Labiatae. Estudos

comprovam a atividade farmacológica deste monoterpeno. No entanto, modificações

estruturais podem diminuir a dose efetiva deste composto. Desta forma, no presente estudo

realizamos uma extensa revisão sistemática que avaliou a atividade anti-inflamatória de

terpenos que sofreram modificações em sua estrutura, através de síntese. Adicionalmente,

sintetizar o propionato de carvacrol (CP), a partir do carvacrol, e avaliar seus possíveis

efeitos antinocicepivo, anti-hiperalgésico e anti-inflamatório. Para construir a revisão, foi

realizada a busca nas bases de dados Scopus, PubMed e Embase, utilizando os descritores

agentes anti-inflamatórios, terpenos e relação estrutura atividade. Já para a parte

experimental, foram utilizados camundongos Swiss machos (25-35 g) com 2 a 3 meses de

idade. Os animais foram divididos em grupos e foram tratados com CP (25, 50 e 100

mg/kg), veículo (solução salina 0,9% + Tween 80 0,2%) ou droga padrão, por via

intraperitoneal (i.p.). O efeito antinociceptivo foi avaliado utilizando o protocolo de

formalina (1%) e o teste da placa quente. A hiperalgesia mecânica foi avaliada após a

administração dos agentes álgicos carragenina (CG; 300 µg/pata), fator de necrose

tumoral-α (TNF-α; 100 pg/pata), prostaglandina E2 (PGE2; 100 ƞg/pata) ou dopamina (DA;

30 µg/pata) utilizando o analgesímetro digital Von Frey. Na avaliação do efeito anti-

inflamatório utilizou-se o teste de pleurisia e edema de pata induzido por CG (1%) em

pletismômetro digital. A citotoxicidade foi avaliada através do método colorimétrico MTT.

Os protocolos experimentais foram aprovados pelo comitê de ética da UFS (CEPA/UFS:

35/12). Os resultados foram expressos como média ± erro padrão da média e as diferenças

entre os grupos foram analisadas por meio do teste de variância ANOVA, uma via ou duas

vias, seguido pelo teste de Tukey ou Bonferroni. Valores de p < 0,05 foram considerados

estatisticamente significantes. Na revisão sistemática foram encontrados 27 artigos sobre

modificação estrutural de terpenos e atividade anti-inflamatória. Na parte experimental, a

administração do CP produziu uma redução significativa (p < 0,01 ou 0,001) no teste da

nocicepção induzida por formalina, em ambas as fases do teste. No teste da placa quente, o

tempo de reação aumentou significativamente nas doses de 50 e 100 mg/kg (p < 0,05; 0,01

ou 0,001). O CP também foi capaz de inibir o desenvolvimento da hiperalgesia mecânica

induzida por todos os agentes testados (p < 0,05; 0,01 ou 0,001). Na avaliação da atividade

anti-inflamatória, o tratamento com CP causou uma diminuição significativa (p < 0,001) no

número total de leucócitos, diminuindo os níveis de TNF-α (p < 0,001), IL-1β (p < 0,05) e

extravasamento de proteínas (p < 0,01). Além disso, o edema de pata induzido por CG

também foi inibido pelo CP (p < 0,05; 0,01 ou 0,001). Desta forma, conclui-se que o CP

possui atividade antinociceptiva, anti-hiperalgésica e anti-inflamatória, provavelmente por

inibição de citocinas. Dessa maneira, a modificação estrutural em terpeno pode ser uma

alternativa interessante para obtenção de moléculas com propriedades farmacológicas.

Palavras-chaves: terpeno, modificação estrutural, carvacrol, propionato de carvacrol,

inflamação, hiperalgesia.

viii

ABSTRACT

SANTANA, M.T. CARVACROL PROPIONATE SYNTHESIS AND STUDY OF ITS

ANTI-HYPERALGESIC AND ANTI-INFLAMMATORY PROPERTIES IN

EXPERIMENTAL PROTOCOLS. Dissertação de Mestrado em Ciências Farmacêuticas,

Universidade Federal de Sergipe, 2013.

Terpenes are naturally occurring compounds obtained from the plants secondary

metabolism. Despite presenting pharmacological effects, structural changes within their

skeleton may increasing their pharmacological activity and attenuate the toxicological

effects. Carvacrol is a phenolic monoterpene present in essential oils from plants belonging

to the Labiatae family. Studies have demonstrated that carvacrol has anti-inflammatory

activity. However, sctructural changes may reduce the effective dose of this monoterpene.

Thus, in this study, we conducted an extensive systematic review evaluating the anti-

inflammatory activity of terpenes that suffered an alteration in its structure through

synthesis and semi-synthesis, synthesize the carvacrol propionate (CP) from the carvacrol

and evaluate its potential antinociceptive, anti-hyperalgesic and anti-inflammatory effects.

To build the revision, it was made the search in Scopus, Embase and PubMed databases,

using the descriptors anti-inflammatory agents, terpenes and structure activity relationship.

In the experimental part, it was used Male Swiss mice (25-35 g) with 2 to 3 months age.

The animals were divided in groups and were treated with CP (25, 50 and 100 mg/kg),

vehicle (saline solution 0.9% + Tween 80 0.2%) or standard drug, intraperitoneally (i.p.).

The antinociceptive effect was evaluated through the formalin (1%) protocol and the hot

plate test. The mechanical hyperalgesia was evaluated through the algic agents injection:

carrageenan (CG; 300 µg/paw), tumor necrosis factor-α (TNF-α; 100 pg/paw),

prostaglandin E2 (PGE2; 100 ng/paw) or dopamine (DA; 30 μg/paw) using a digital

analgesimeter (von Frey). To assess the anti-inflammatory effect, it was used the pleurisy and

paw edema induced by GC (1 %) in digital plethysmometer. The cytotoxicity of CP was

evaluated by the MTT colorimetric method. The experimental protocols were approved by

the UFS ethics committee (CEPA/UFS: 35/12). The results are expressed as mean ± SEM

and differences between groups were analyzed by one-way or two-way ANOVA test

followed by Tukey or Bonferroni tests. Values of p < 0.05 were considered statistically

significant. In systematic review, 27 papers were found concerning about terpenes

structural modification and the evaluation of their anti-inflammatory activity. In the

experimental part, the administration of CP produced a significant decrease (p < 0.01 and

0.001) in the test formalin-induced nociceptive in both phases of the test. In the hot plate

test, the reaction time increased significantly at doses 50 and 100 mg/kg (p < 0.05, 0.01

and 0.001). CP inhibited the development of mechanical hyperalgesic induced by all agents

tested (p < 0.05, 0.01 and 0.001). In the evaluation of anti-inflammatory activity, the

treatment with CP was able to decrease significantly the leukocyte recruitment (p < 0.001),

the TNF-α (p < 0.001), the IL-1β (p < 0.05) and protein leakage (p < 0.01). In addition, the

paw edema induced by CG in mice was inhibited significantly by CP (p < 0.05, 0.01 and

0.001). Thus, it is concluded that the CP attenuates nociception, mechanical hyperalgesia

and inflammation, through an inhibition of cytokines. Therefore, structural modification

terpene can be an interesting alternative for obtaining molecules with pharmacological

properties.

Key-words: terpene, structural modification, carvacrol, carvacrol propionate,

inflammation, hyperalgesia.

ix

SUMÁRIO

1.0 INTRODUÇÃO ................................................................................................................ 2

REFERÊNCIAS....................................................................................................................... 6

2.0 OBJETIVOS ..................................................................................................................... 9

2.1 OBJETIVO GERAL .......................................................................................................... 10

2.2 OBJETIVOS ESPECÍFICOS............................................................................................. 10

3.0 DESENVOLVIMENTO....................................................................................................... 11

3.1 CAPÍTULO 1 - STRUCTURE-ACTIVITY RELATIONSHIP OF TERPENES WITH

ANTI-INFLAMMATORY PROFILE – A SYSTEMATIC REVIEW………….…………………...

12

3.2 CAPÍTULO 2- SYNTHESIS AND PHARMACOLOGICAL EVALUATION OF

CARVACROL PROPIONATE………………………………………………………………….......

55

4.0 CONCLUSÃO ................................................................................................................... 88

ANEXOS ................................................................................................................................. 89

x

ÍNDICE DE FIGURAS

Capítulo 1 - Structure-activity relationship of terpenes with anti-inflammatory profile – a systematic review

Figure 1 Flowchart of included studies …………………..………………….…..…..38

Figure 2 Structures of terpene derivatives..............…………………………………38

Capítulo 2 - Synthesis and pharmacological evaluation of carvacrol propionate

Figure 1. Sythesis reaction of the carvacrol propionate (CP) from the reagents carvacrol, triethylamine and propionile chloride……………………………………...81

Figure 2. Effects of carvacrol proprionate (CP; 25, 50 or 100 mg/kg, i.p.) or morphine (MOR, 3 mg/kg; i.p.) on formalin-induced nociceptive behavior were administered intraperitoneally 0.5 hr before formalina injection. (panel A) First phase (0-5 min.) and (panel B) second phase (15-30 min.) of the formalin test. Values represent mean ± S.E.M. (n = 6, per group). **p < 0.01 and ***p < 0.001 versus control (one-way ANOVA followed by Tukey’stest)……..…………………..81

Figure 3. Effect of acute administration of vehicle, carvacrol propionate (CP; 25, 50 or 100 mg/kg, i.p.), indomethacin (IND, 10 mg/kg, i.p.) or dipyrone (DIP, 60 mg/kg, i.p.) on mechanical hypernociception induced by carrageenan (A), TNF-α (B), PGE2 (C) and dopamine (D). Each point represents the mean ± S.E.M. of the paw withdrawal threshold (in grams) to tactile stimulation of the left hind paw. * p < 0.05, **p < 0.01 and ***p < 0.001 vs. control group (ANOVA followed by Tukey test)...............................................................................................................……..82

Figure 4. Effect of acute administration of vehicle, carvacrol propionate (CP; 25, 50 or 100 mg/kg, i.p.) or indomethacin (IND, 10 mg/kg; i.p.) on the inflammation by carrageenan in mice pleurisy. The analyses were performed 4 h after carrageenan injection (300 μg/cavity) to evaluate the recruitment of total leukocytes (A), neutrophils (B). Data were expressed as mean ± SEM, for a minimum of six animals. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the control group (vehicle) (ANOVA followed by Tukey test)…………………………………………...83

xi

Figure 5. Effect of vehicle, carvacrol propionate (CP; 1, 10, 100, 250 or 500 µg/mL, in vitro) on murine peritoneal macrophages (2.5×105 cells). The percentage of viability was determined in relation to controls. Data were expressed as mean ± SEM. ** p < 0.01 compared with the control group (vehicle) (ANOVA followed by Tukey test)…….................................................................................................…..83

Figure 6. Effect of acute administration of vehicle, carvacrol propionate (CP; 25, 50 or 100 mg/kg, i.p.) or indomethacin (IND, 10 mg/kg; i.p.) on the inflammation by carrageenan in mice pleurisy. The analyses were performed 4 h after carrageenan injection (300 μg/cavity) to evaluate to assess tumor necrosis factor-alpha (TNF-α) (A), and interleukin-1β (IL-1β) levels (B), and total protein (C). Data were expressed as mean ± SEM, for a minimum of six animals. * p < 0.05, ** p < 0.01, and *** p <0.001 compared with the control group (vehicle) (ANOVA followed by Tukey test)………………………………………………………………….……...…….84

Figure 7. Effect of acute administration of vehicle, carvacrol proprionate (CP; 25, 50 or 100 mg/kg, i.p.) or indomethacin (IND, 10 mg/kg; i.p.) on edema induced by carrageenan. Each point represents the mean±SEM of the paw volume (in milliliter, panel A) or the area under curve (AUC) from 0 to 6 h (panel B). *p < 0.05, **p < 0.01 and ***p < 0.001 vs. control group (ANOVA followed by Tukey test)……………………………………………………………..…………..…….….......85

xii

ÍNDICE DE TABELAS

Capítulo 1 - Structure-activity relationship of terpenes with anti-inflammatory profile – a systematic review

Table 1 Description of the modification chemical of the terpenes and

pharmacological aspects of the of the studies included in systematic review........50

Capítulo 2 - Synthesis and pharmacological evaluation of carvacrol

propionate

Table 1. Effect of CP (25, 50, or 100 mg/kg; i.p.) or MOR (3.0 mg/kg; i.p.) on the hot plate test in mice…………………………………………………………………….86

xiii

LISTA DE ABREVIATURAS

AINES – Anti-inflamatório não esteroidais

AMPc – Adenosina monofosfato cíclico

Cg – Carragenina

COX – Enzima ciclo-oxigenase

CP – Propionato de Carvacrol

DA - Dopamina

DIP – Dipirona

IL – Interleucina

IND – Indometacina

MOR – Morfina

MTT - Brometo de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólio

NF-kB – Fator Nuclear Kappa B

NO – Óxido Nítrico

PGE2 - Prostaglandina E2

PPAR – Receptor ativados por proliferador de peroxisomo

TNF-α – Fator de Necrose Tumoral alfa

1

1.0 INTRODUÇÃO

2

1.0 INTRODUÇÃO

O processo inflamatório é a resposta do organismo a diferentes estímulos,

incluindo danos mecânicos, físicos, químicos e biológicos (Gregory et al.,

2008). De forma controlada, é uma resposta benéfica que protege o organismo

contra os agentes invasores, uma vez que atenua uma infecção contribuindo

até o retorno da homeostase (Cotran et al., 2006). No entanto, devido à

resistência do patógeno, a inflamação pode se tornar crônica, levando a um

aumento do número de mediadores inflamatórios que conduz a efeito nocivo

ao organismo (Medzhitov, 2008).

As características do processo inflamatório incluem uma complexa cascata

de eventos bioquímicos e celulares, que envolve extravasamento de líquido,

migração celular, produção de mediadores pró-inflamatórios e sensibilização

de nociceptores (Becker, 1983). Estas por sua vez, geram a sintomatologia

característica da inflamação, conhecida pelos cinco sinais cardinais: eritema,

calor, rubor, dor e a perda da função (Medzhitov, 2008).

O aumento da sensibilidade a estímulos dolorosos, conhecido como

hiperalgesia, é uma característica marcante da inflamação. Mediadores

inflamatórios, liberados por células inflamatórias como, tais como, citocinas

(Interleucina-1β, Fator de Necrose Tumoral-α) estimulam a produção de

metabólitos da enzima ciclo-oxigenase (COX) e aminas simpatomiméticas.

Estes contribuem para aumentar a sensibilização dos receptores nociceptivos

(Woolf e Ma, 2007).

Provavelmente, devido à interação destes mediadores a canais iônicos de

membrana, tipo voltagem-dependente ou receptores da membrana acoplados

a proteínas regulatórias denominadas de proteínas G (Ferreira, 1995). Ambos

receptores quando ativados elevam as concentrações de adenosina

monofosfato cíclico (AMPc) e cálcio intracelular, contribuindo para diminuição

do limiar de excitabilidade neural (Rocha et al., 2007).

Atualmente, o manejo terapêutico para condições inflamatórias e

dolorosas, foca na cascata da inflamação (Carvalho e Lemônica, 1998). A

primeira escolha para o tratamento inclui os anti-inflamatórios não esteroidais

(AINES), que são fármacos inibidores da COX, consequentemente bloqueiam

a formação de mediadores finais, tais como, prostaglandinas (PGE2), os

3

medicamentos desta classe incluem a aspirina, indometacina, diclofenaco

(Rao et al., 2003). A segunda opção de tratamento é impedir o

desenvolvimento da hiperalgesia, através do mecanismo de dessensibilização,

consequentemente restaurando o limiar do nociceptor, pode-se destacar a

morfina e a dipirona (Rodrigues e Duarte, 2000; Reis e Rocha, 2006).

No entanto, estes medicamentos provocam efeitos adversos como, lesão

gástrica, nefrotoxidade, náuseas e efeito tolerância (Vane et al., 1998; Furlan

et al., 2006). Logo, existe a necessidade clínica para a procura de novas

drogas anti-inflamatórias. Esta busca se dá através de melhorias das práticas

em investigações pré-clínicas e o refinamento em modelos animais que

mimetizem as condições inflamatórias (Knowles, 2013).

Dessa maneira, alternativas farmacológicas que apresentem alta eficácia

no tratamento e menos efeitos indesejáveis são necessárias (Wang et al.,

2013). Em resposta à demanda de novos medicamentos para o tratamento da

dor inflamatória, os produtos naturais e derivados representam uma

ferramenta farmacológica de extrema importância. Uma vez que apresentam

uma grande diversidade e complexidade de estrutura química, o que não é

visto nos compostos puramente sintéticos. Por isso, é de extrema importância

a descoberta de novos fármacos para o tratamento de diversas doenças que

acometem a população (Gautam e Jachak, 2009).

Além disso, de acordo com estudo de Porto et al., (2009), a modificação

estrutural realizada em produtos naturais originando uma nova molécula pode

apresentar atividades promissoras, visto como uma forma interessante de

obtenção de novas estruturas, com a possibilidade de aprimoramento da sua

atividade.

Logo, o estudo da relação estrutura-atividade de produtos naturais é

considerado, atualmente, uma ferramenta fundamental no planejamento de

novos protótipos de fármacos (Vechia et al., 2009). O fato é que, uma pequena

modificação na estrutura pode conduzir a uma alteração na atividade biológica

(Guha, 2012), permitindo que químicos possam realizar substituições

especificas que melhorem as propriedades da molécula, como lipofilicidade,

esta que contribui com a biodisponibilidade da droga no organismo (Martin et

al., 2002).

4

Dessa maneira, realizar modificações na estrutura de um composto ativo,

pode aumentar a sua eficácia e também a seletividade, diminuindo a

toxicidade. Portanto, o interesse da comunidade científica pelos produtos

naturais tem como objetivo, descobrir novas entidades químicas ativas,

passíveis de modificações que representem potencialidades terapêuticas,

contribuindo assim para a prevenção e/ou tratamento de determinadas

doenças (Dias et al., 2012).

Muitos fármacos disponíveis atualmente foram obtidos sinteticamente,

baseados em estruturas naturais ativas (Bauer e Brönstrup, 2013). Em se

tratando do efeito analgésico, um grande exemplo de sucesso terapêutico na

modificação estrutural de um composto natural é o ácido acetilsalicílico,

primeiro produto sintético para fins terapêuticos, obtido a partir de um

glicosídeo natural, salicina, identificado como princípio ativo de Salix sp

(Barreiro e Bozani, 2009).

A morfina também merece destaque, uma vez que também foi

originalmente isolada da Papaver somniferum, e inspirou a descoberta

posterior dos derivados 4-fenil-piperidínicos, a meperidina, destacando-se pela

reduzida propriedade indutora de tolerância quando comparada ao produto

natural morfina (Barreiro e Manssour, 2008).

Dentro deste contexto, os monoterpenos, representantes de uma classe de

compostos químicos chamados de terpenos, constituintes dos óleos

essenciais de plantas, são ricos em substâncias químicas com atividade

biológica (Barbosa-Filho et al., 2006). Apesar de possuir uma estrutura

simples, uma vez que apresentam duas unidades isoprênicas, apresentam

diversas atividades biológicas (Las Heras et al., 2003)

Por isso, sua importância para a comunidade cientifica, já que existem

diversos estudos que comprovam seu efeito farmacológico (Quintans-Júnior et

al., 2010; Batista et al., 2010; Riella et al., 2012). Dentre os monoterpenos,

pode-se destacar o carvacrol, presente em óleos essenciais de plantas

pertencentes à família Labiatae. Nos últimos anos, estudos comprovam que o

carvacrol tem efeito anti-inflamatório provavelmente por inibição de

mediadores como PGE2, IL-1β e TNF-α. (Guimarães et al., 2012; Lima et al.,

2013). Também já foi comprovada a inibição da enzima ciclo-oxigenase-2

5

(Landa et al., 2009), além de estimular os receptores ativados por proliferador

de peroxisomo (PPAR) (Hotta et al., 2010). No entanto, os estudos

demonstram a atividade deste monoterpeno em doses relativamente altas.

Por isso, como alternativa de diminuir a dose efetiva de compostos ativos,

modificações estruturais podem ser propostas, para a obtenção de uma nova

molécula ativa (Carvalho et al., 2003). Alguns estudos já comprovam a eficácia

de modificações estruturais em plantas medicinais, obtendo derivados

sintéticos (Newman et al., 2003). Podem ser citados a di-hidrocarvona, um

derivado sintético da carvona que apresentou propriedade anti-inflamatória

(De Souza et al., 2010) e antinociceptiva (Oliveira et al., 2008). Análogos

sintéticos da rotundifolona demonstraram efeito antinociceptivo significativo

(De Sousa et al., 2007).

Uma estrutura interessante, que contribui com potencial efeito anti-

inflamatório é a classe dos propionatos, visto que na clínica já se utilizam o

proprionato de clobetasol, propionato de fluticasona e dipropionato de

betametasona para condições inflamatórias (Menter et al., 2012; Ynson et al.,

2013). No entanto, como são anti-inflamatórios esteroidais, o seu uso é

limitado, devido às reações adversas associadas (Stuetz et al., 2001).

Baseado na literatura, e levando-se em consideração que a modificação

estrutural de produtos naturais é uma fonte importante para a obtenção de

moléculas biologicamente ativas, uma vez que diversos medicamentos

utilizados no tratamento de várias doenças são oriundos desta forma. Estudos

científicos voltados à análise da efetividade das modificações estruturais em

plantas medicinais são escassos, tornam-se necessárias pesquisas voltadas

para a descoberta de uma nova estrutura com potencial terapêutico.

Dessa maneira, visando atender a necessidade no desenvolvimento de

fármacos anti-inflamatórios com menores efeitos colaterais e considerando a

possibilidade de modificações estruturais em monoterpenos resultarem em

novas entidades químicas com propriedade analgésicas (De Souza et al.,

2007), este trabalho teve como foco realizar uma revisão sistemática,

buscando verificar se, modificação estrutural em terpenos melhora a atividade

anti-inflamatória. Adicionalmente sintetizar o propionato de carvacrol (CP) e

avaliar os possíveis efeitos analgésico e anti-inflamatório deste composto.

6

REFERÊNCIAS

Barbosa-Filho JM, Medeiros KCP, Diniz MFFM, Batista LM, Athayde-Filho PF, Silva MS, et al. Natural products inhibitors of the enzyme acetylcholinesterase. J Braz Pharmacogn 2006; 16: 258-285. Barreiro EJ, Bolzani VS. Biodiversity: potential source for drug discovery. Quim Nova 2009; 32(3): 679-688. Barreiro EJ, Manssour CAM. Química Medicinal: As Bases Moleculares da Ação dos Fármacos Ed. Artmed 1ª ed. Porto Alegre 2008: p.161-178. Batista PA, Werner MFP, Oliveira EC, Burgos L, Pereira P, Brum LFS, et al. The antinociceptive effect of (−)-linalool in models of chronic inflammatory and neuropathic hypersensitivity in mice. J Pain 2010; 11: 1222-1229. Bauer A, Brönstrup M. Industrial natural product chemistry for drug discovery and development. Nat Prod Rep 2013 DOI: 10.1039/c3np70058e. Becker EL. Chemotactic fators of inflamation. Trends Pharmacol Sci 1983; 4(5): 223-225. Carvalho I, Pupo MT, Borges ADL, Bernardes LSC. Introduction to molecular modeling of drugs in the medicinal chemistry experimental course. Quim Nova 2003; 26(3): 428-438. Carvalho WA, Lemônica L. Molecular and Cellular Mechanisms of Inflammatory Pain. Peripheral Modulation and Therapeutic Advances. Rev Bras Anestesiol 1998; 48(2):137-158. Cotran RS, Kumar V, Collins T. Patologia estrutural e funcional Ed. Guanabara Koogan 7ª ed. Rio de Janeiro 2006: pp. 29. De Sousa DP, Camargo EA, Oliveira FS, de Almeida RN. Anti-inflammatory activity of hydroxydihydrocarvone. Z Naturforsch C 2010; 65(9-10): 543-550. De Sousa DP, Júnior EV, Oliveira FS, De Almeida RN, Nunes XP, Barbosa-Filho JM. Antinociceptive activity of structural analogues of rotundifolone: structure-activity relationship. Z Naturforsch C 2007; 62(1-2): 39-42. Dias DA, Urban S, Roessner U. A Historical Overview of Natural Products in Drug Discovery. Metabolites 2012; 2: 303-336. Ferreira SH. Hiperalgesia inflamatória, óxido nítrico y control periférico del dolor. Rev Lati Ame Dolor 1995; 1: 6-17. Furlan AD, Sandoval JA, Mailis-Gagnon A, Tunks E. Opioids for chronic noncancer pain: a meta analysis of effectiveness and side effects. Can Med Assoc J 2006; 174(11): 1589-1594.

7

Gautam R, Jachak SM. Recent Developments in Anti-Inflammatory Natural Products. Med Res Rev 2009; 29(5): 767-820. Gregory M, Barton A. Calculated response: control of inflammation by the innate immune system. J Clin Invest 2008; 118: 413-420. Guha R Exploring Structure-Activity Data Using the Landscape Paradigm. Wiley Interdiscip Rev Comput Mol Sci. 2012; 2(6): 1-18. Guimaraes AG, Xavier MA, de Santana MT, Camargo EA, Santos CA, Brito FA, et al. Carvacrol attenuates mechanical hypernociception and inflammatory response. N-S Arch Pharmacol 2012; 385(3): 253-63. Hotta M, Nakata R, Katsukawa M, Hori K, Takahashi, Inoue H. Carvacrol, a component of thyme oil, activates PPAR alpha and gamma, and suppresses COX-2 expression. J Lipid Res 2010; 51: 132-139. Knowles RG. Development of anti-Inflammatory drugs – the research and development process. Basic Clin Pharmacol Toxicol 2013; doi: 10.1111/bcpt.12130. Landa P, Kokoska L, Pribylova M, Vanek T, Marsik P. In vitro anti-inflammatory activity of carvacrol: Inhibitory effect on COX-2 catalyzed prostaglandin E(2) biosynthesis. Arch Pharm Res 2009; 32(1):75-78. Las Heras B, Rodriguez B, Bosca L, Villar AM. Terpenoids: sources, structure elucidation and therapeutic potential in inflammation. Curr Top Med Chem 2003; 3(2): 171-185. Lima Mda S, Quintans-Junior LJ, De Santana WA, Martins Kaneto C, Pereira Soares MB, Villarreal CF. Anti-inflammatory effects of carvacrol: evidence for a key role of interleukin-10. Eur J Pharmacol 2013; 699(1-3): 112-117. Martin YC, Kofron JL, Traphagen LM. Do Structurally Similar Molecules Have Similar Biological Activity?. J Med Chem 2002; 45: 4350-4358 Medzhitov R. Origin and physiological roles of inflammation. Nature 2008; 454: 428-435. Menter MA, Caveney SW, Gottschalk RW. Impact of clobetasol propionate 0.05% spray on health-related quality of life in patients with plaque psoriasis. J Drugs Dermatol 2012; 11(11): 1348-1354. Newman DJ, Cragg GM, Snader KM. Natural products as sources of new drugs over the period 1981-2002. J Nat Prod 2003; 66:1022-1037. Oliveira FS, De Sousa DP, de Almeida RN. Antinociceptive effect of hydroxydihydrocarvone. Biol & Pharm Bull 2008; 31(4): 588-591.

8

Porto TS, Furtado NAJC, Heleno VCG, Martins CHG, Da Costa FB, Severiano ME, et al. Antimicrobial ent-pimarane diterpenes from Viguiera are naria against Gram positive bactéria. Fitoterapia 2009; 80: 432-436. Quintans-Junior LJ, Melo MS, De Sousa DP, Araujo AA, Onofre AC, Gelain DP, et al. Antinociceptive effects of citronellal in formalin-, capsaicin-, and glutamate-induced orofacial nociception in rodents and its action on nerve excitability. J Orofac Pain 2010; 24(3): 305-312. Rao P, Knaus EE. Evolution of nonsteroidal anti-inflammatory drugs (NSAIDs): cyclooxygenase (COX) inhibition and beyond. J Pharm Sci 2008;11(2): 81-110 Reis FJ, Rocha NP. Long term analgesic effect of dipyrone on the persistent hyperalgesia induced by chronic constriction injury of sciatic nerve in rats: involviment of nitric oxide. Braz J Pharm Sci 2006; 42(4): 513-522. Riella KR, Marinho RR, Santos JS, Pereira-Filho RN, Cardoso JC, Albuquerque-Junior RLC, et al. Anti-inflammatory and cicatrizing activities of thymol, a monoterpene of the essential oil from Lippia gracilis, in rodents. J Ethnopharmacol 2012; 143: 656-663. Rocha APC, Kraychete DC, Lemonica L, Carvalho LR, Barros GAM, Garcia JBS, et al. Pain: Current Aspects on Peripheral and Central Sensitization. Rev Bras Anestesiol 2007; 57(1): 94-105. Rodrigues AR, Duarte IDG. The peripheral antinociceptive effect induced by morphine is associated with ATP-sensitive K+channels. Br J Pharmacol 2000; 127: 110-114. Stuetz A, Grassberger M, Meingassner JG. Pimecrolimus (Elidel, SDZ ASM 981)-preclinical pharmacologic profile and skin selectivity. Semin Cutan Med Surg. 2001; 20(4): 233-241. Vane JR, Bakhle YS, Botting RM. Cyclooxygenases 1 and 2. Annu Rev Pharmacol Toxicol 1998; 38: 97-120. Vechia LD, Gnoatto SCB Gosmann G. Oleanane and ursane derivatives and their importance on the discovery of potential antitumour, antiinflammatory and antioxidant drugs. Quim Nova 2009; 32(5): 1245-1252. Wang Q, Kuang H, Su Y, Sun Y, Feng J, Guo R, Chan K. Naturally derived anti-inflammatory compounds from Chinese medicinal plants. J Ethnopharmacol 2013; 146: 9-39. Woolf CJ, Ma Q. Nociceptors-noxious stimulus detectors. Neuron 2007; 55: 353-364. Ynson ML, Forouhar F, Vaziri H. Case report and review of esophageal lichen planus treated with fluticasone. World J Gastroenterol 2013; 19(10):1652-1656.

9

2.0 OBJETIVOS

10

2.0 OBJETIVOS

2.1 GERAL

Sintetizar e determinar a estrutura do propionato do carvacrol (CP) e avaliar

seus efeitos antinociceptivo, anti-hiperalgésico e anti-inflamatório em protocolos

experimentais.

2.2. ESPECÍFICOS

Realizar um levantamento bibliográfico buscando a construção de uma

revisão sistemática sobre a relação estrutura atividade de terpenos com

efeito anti-inflamatório;

Sintetizar e determinar a estrutura do CP;

Avaliar a ação antinociceptiva do CP;

Verificar a atividade do CP na hiperalgesia mecânica induzida por diversos

agentes;

Avaliar o efeito do CP em modelos inflamatórios e quantificar a produção

de mediadores pró-inflamatórios;

Analizar traços de citotoxicidade do CP;

Verificar a possível interferência do CP sobre a coordenação motora dos

animais.

11

3.0 DESENVOLVIMENTO

12

3.1 CAPÍTULO 1

STRUCTURE-ACTIVITY RELATIONSHIP OF TERPENES

WITH ANTI-INFLAMMATORY PROFILE – A SYSTEMATIC

REVIEW

Artigo publicado ao periódico:

Basic & Clinical Pharmacology & Toxicology

Fator de impacto no Journal Citation Reports® (JCR):

2.124

13

14

STRUCTURE-ACTIVITY RELATIONSHIP OF TERPENES WITH ANTI-

INFLAMMATORY PROFILE – A SYSTEMATIC REVIEW

Marilia Trindade de Santana Souza1, Jackson Roberto Guedes da Silva Almeida

2, Adriano

Antunes de Souza Araujo3, Marcelo Cavalcante Duarte

3 and Lucindo José Quintans Júnior

1,*

1Department of Physiology, Federal University of Sergipe, São Cristovão, SE, Brazil

2 College of Pharmaceutical Sciences, Federal University of São Francisco Valley, Petrolina,

PE, Brazil

3Department of pharmacy, Federal University of Sergipe, São Cristovão, SE, Brazil

*Corresponding author: Laboratório de Farmacologia Pré-Clínica, Universidade Federal de

Sergipe-UFS, Av. Marechal Rondom, s/n, São Cristóvão, Sergipe-Brazil. Tel.: +55-79-

21056645; fax: +55-79-3212-6640. E-mail address: lucindojr@gmail.com;

lucindo@pq.cnpq.br

15

Abstract: Inflammation is a complex biological response that has no treatment without side

effects. Because of this, natural products have been the subject of incessant studies, among

which the class of terpenes stands out. They have been the source of study for the

development of anti-inflammatory drugs, once their chemical diversity is well suited to

provide skeleton for future anti-inflammatory drugs. This systematic review reports the

studies present in the literature that evaluate the anti-inflammatory activity of terpenoids

suffering any change in their structures, assessing whether these changes also brought changes

in their effects. The search terms anti-inflammatory agents, terpenes, structure-activity

relationship were used to retrieve English language articles in SCOPUS, PUBMED and

EMBASE published between January 2002 and August 2013. Twenty-seven papers were

found concerning the structural modification of terpenes with evaluation of the anti-

inflammatory activity. The data reviewed here suggest that modified terpenes are an

interesting tool for the development of new anti-inflammatory drugs.

Keywords: Anti-inflammatory agents, terpenes, structure-activity relationship.

16

INTRODUCTION

The word ‘inflammation’ comes from the Latin inflammare (to set on fire) and it is

defined as a complex biological response of vascular tissues against aggressive agents,

involving a cascade of biochemical events comprising the local vascular system, the immune

system and different cell types found in the injured tissue [1,2].

For the treatment of various inflammatory diseases, the nonsteroidal anti-inflammatory

drugs (NSAIDs) are most widely prescribed, but the gastrointestinal, renal and cardiovascular

toxicity associated with common NSAIDs limits their usefulness [3]. Because of this, the

potential therapeutic evaluation of the medicinal plants has been the subject of incessant

studies, which have been proven pharmacological actions, such as the anti-inflammatory, of

some plants and their constituents, including the terpenoids [4].

Terpenoids, which make up a very large family of natural products, contain more than

50,000 structurally diverse compounds, which are categorized by number of C5 isoprene units

[5]. Terpenoids have been described with important biological activities, such as analgesic [6,

7], anticonvulsant [8] and cardiovascular [9]. Additionally, anti-inflammatory activity of some

terpenoids is described in the literature, such as: β-caryophyllene [10], citral [11], α-pinene

[12], citronellal [13], limonene [14] and surgiol [15].

Despite the existing technology in organic chemistry for the synthesis of a new drug,

the natural products, including terpenes, serve as a source of raw material for innovative drug

discovery [16], once the chemical diversity of terpenes is well suited to provide skeleton for

future drugs [17]. Thus, in an attempt to improve the efficacy/safety profile of new anti-

inflammatory drugs, including those of natural origin, the structural-activity relationship has

been extensively studied, taking into account up-to-date knowledge on the mechanism of

inflammation [18, 19]. Despite its importance, there are no reviews on the anti-inflammatory

activity of structurally-modified terpene.

17

In this context, the present study aimed to analyze, through a systematic review, the

studies present in the literature that evaluate the anti-inflammatory activity of terpenoids that

suffered any change in their structures, assessing whether these changes also brought changes

in their effects.

METHODS

A systematic review was carried out through a literature search performed in August

2013 and included articles published over a period of 10 years (January 2002 to August

2013). This literature search was performed through specialized databases (PUBMED,

SCOPUS and EMBASE) using different combinations of the following keywords: terpenes,

anti-inflammatory agents, structure-activity relationship. The manuscript selection was based

on the inclusion criteria: articles published in English and articles with keywords in the title,

abstract or full text, as well as studies with isolated terpenes for further structural

modifications. Articles conducted with structure-activity relationship isolated from plants

were excluded.

For the selection of the manuscripts, two independent investigators (MTSS and LJQJ)

first selected the articles according to the title, then to the abstract and then through an

analysis of the full-text publication. Any disagreement was resolved through a consensus

between them. The resulting articles were manually reviewed with the goal of identifying and

excluding the works that did not fit the criteria described above.

RESULTS AND DISCUSSION

This review searched for structural modifications in terpenes which enhanced the anti-

inflammatory activity in the last ten years. The primary search identified 762 articles, with

445 from PUBMED, 13 from SCOPUS and 304 from EMBASE. However, out of this total, 7

were indexed in two or more databases and were considered only once, resulting in 755

18

articles or referred to studies. After the initial screening of the titles, abstracts, full text and

time of publication, 27 articles were selected and the others did not meet the inclusion criteria

(n = 728). We excluded studies that evaluated the structure-activity relationship of only

compounds isolated from plants or papers that were not within the limits of the year (January

2002 and August 2013). A flowchart illustrating the progressive study selection and numbers

at each stage is shown in the Figure 1.

Structural modification of natural products showed promising activities that must be

seen as an interesting source of new structures, with the possibility of presenting a better

biological activity [20]. Table 1 and Fig. 2 show the chemical modification and

pharmacological aspects of the terpenes identified by this systematic review.

It was possible to verify that structural changes in terpene compounds are common in

order to improve the anti-inflammatory activity. The most used protocol was in vitro tests,

with only a few in vivo tests and topical administration.

Monoterpene

Isoegomaketone (IK)

This monoterpene is the main essential oil component of Perilla frutescens. In an

attempt to enhance the activity, we proposed a chemical modification in the IK, focusing on

the aromatic heterocyclic ring. It was carried out to improve the suppressive effects on the

production of NO, MCP-1 and IL-6, important mediators in the inflammatory process, which

were evaluated through the regulation of the NF-κB and AP-1 transcriptional activation [21].

The IK and its five derivatives were able shown to inhibit the NO, MCP-1 and IL-6

formation by the LPS-induced inflammatory responses, that it was investigated in RAW 264.7

mouse macrophage cell line. Besides, it would inhibit the expression of these genes through

the suppression of NF-κB or AP-1 activation. Among the synthesized derivatives, we could

19

check that the introduction of a methyl group at the 5-position furan ring in the IK improved

threefold the inhibitory activities towards NO and MCP-1 production. Furthermore, a

significant suppression of NF-κB and AP-1 DNA binding activities was shown for this

derivative [21].

Sesquiterpene lactones

Pseudoguaianolides, psilostachyin, parthenin and coronopilin are sesquiterpenes

lactones, in other words, have 3 isoprene units merged into a lactone ring. These compounds

are found in the species Parthenium hysterophorus, Ambrosia psilostachya, Parthenium

hysterophorus, respectively. The modifications of these sesquiterpenes form the type:

acetylation at C-1 and, subsequently, inserted a propionate and butyrate group. The parthenin

generates a library of analogues of the type: δ-valerolactones, spirolactone, azaspiro lactones

and butenolide. These were evaluated as to their anti-inflammatory potential through in vitro

TNF-α, IL-1β and IL-6 expression in murine neutrophils [22].

Chib et al [22], interestingly, found that the structural modification improved the

activity of parent molecules, since azaspiro lactones and butenolide analogues displayed

maximum inhibitory effect on TNF-α cytokine secretion. Moreover, they suppressed the

extracellular IL-1β expression level in LPS-activated neutrophils at dose level of 1 µg/ml and

also suppressed the extracellular IL-6 expression at dose level of 1 µg/ml, even though the

inhibition of expression was not significant.

Despite of the fact that the α-methylene-γ-lactones are required for the activity of

sesquiterpene lactones, other steric requisites must be fulfilled [23]. In this case, the insertion

of the azaspiro and butenolide contributed to improve the anti-inflammatory activity.

Parthenolide

20

Another type of sesquiterpene lactone present in the species Tanacetum parthenium is

the parthenolide. Changes in the parthenolide skeleton comprised compounds with different

structure types, such as: guaianolides, pseudoguaianolide, germacrolides, melampolides,

heliangolides and 4,5-dihydrogermacranolides. These changes were proposed by Neukirch

and collaborators [24] in order to improve the IL-8 chemotaxis.

Once this modification occurred, the bicyclic compounds derived from acidic

treatment of parthenolide inhibited the chemotaxis more than did the parthenolide substrate.

In fact, the modest structural changes have marked influence on the migration of neutrophils

[25], demonstrating the α-methylene γ-lactone plays an important role in anti-inflammatory

effect.

However, we did not discard the addition of another structure, since the simple α-

methylene-γ-lactones caused minimal anti-inflammatory activity, which means that for

pharmacological activity, other steric requisites must be considered [23].

Sesquiterpene hydroquinones/quinones

Bolinaquinone

Bolinaquinone is a hydroquinones/quinones sesquiterpene belonging to one class of

marine sponge metabolites, which have received considerable attention due to their varied

biological activities [26]. Aiming at the improvement of the bolinaquinone activity, we

proposed structural changes of the basic molecule variations in the aromatic system and

evaluated the inhibition of PGE2 production in the LPS-treated RAW 264.7 cells [27].

Remarkably, the (4A) inhibitor showed good ability in reducing LPS-induced PGE2

release with potency degrees better than the parent compound, the bolinaquinone.

Curiously, the analogues lack the methyl spacer; in other words, variations in the

aromatic system directly attached to the quinone ring were inactive compounds, which

21

suggested that the linker between the hydrophobic pocket and quinone ring is essential for the

activity [27].

Avarol

Avarol is a marine sesquiterpenoid hydroquinone with interesting pharmacological

properties. Because it is a molecule able of modifications, Amigó and collaborators [28]

undertook the synthesis of the avarol ester derivatives, avarol oxidation and amino

derivatives. We evaluated as potential antipsoriatic agents by the inhibition of superoxide

generation in activated human neutrophils or reduction of cell proliferation and the PGE2

generation in the cultured human keratinocyte HaCaT cell line.

According to Amigó and collaborators [28], the Avarol-3’-thiosalicylate (5A) showed

better anti-inflammatory properties as antioxidant and inhibitor of PGE2 release compared

with the avarol. This result, in summary, could be related to the presence of a thiosalicylic

function at the hydroquinone moiety, which could act through cyclooxygenase (COX)

inhibition, in a manner similar to nonsteroidal anti-inflammatory drugs (NSAIDs) [28].

Furthermore, (5A) derivative inhibits both in vivo and in vitro mediators related to the

inflammatory response. Its action mechanism is related to the inhibition of NF-kB activation

and can be mediated by the down-regulation of intracellular signal-transduction pathways

influenced by ROS, TNF-α and arachidonic acid metabolism [29].

Siphonodictyal

Siphonodictyal is a sesquiterpene belonging to the hydroquinone sesquiterpene class.

Laube and collaborators [30] demonstrated the similar structural synthesis of the

sesquiterpene quinones and hydroquinones from the siphonodictyal tested for their anti-

inflammatory activities.

22

It was found that cyclohexadienone and sesquiterpene o-benzoquinone derivatives

showed a very good inhibition of 3α-hydroxysteroid dehydrogenase (3α-HSD), comparable

with the indomethacin [30]. The 3α-HSD is a key enzyme in the inflammatory cascade

involved in the glucocorticoids metabolism. Thus, it is inhibited by the major nonsteroidal

and steroidal agent types [31]. Therefore, the 3α-HSD inhibition can be used in an assay in

search for anti-inflammatory drugs.

Abscisic acid

The abscisic acid (ABA) is a phytohormone sesquiterpene. It has been showed that it

stimulates several functions of human granulocytes phagocytosis, reactive oxygen species,

nitric oxide production and chemotaxis. Aiming to improve its activity, Grozio and

collaborators [32] synthesized an ABA analog compound, the (7A), evaluating its anti-

inflammatory properties on in vitro human granulocytes and monocytes through its ability to

compete with ABA for binding to cell membranes and to the recently identified human ABA

receptor.

Grozio et al [32] showed that ABA analog has higher affinity than ABA for binding to

granulocyte membranes and inhibiting chemotaxis, phagocytosis, ROS and PGE2 production

by human granulocytes.

Diterpenes

Andrographolide

Andrographolide is a bicyclic diterpenoid lactone isolated from the Andrographis

paniculata (Burm. f) leaves. The novel synthesis of derivatives from andrographolide to

screen for more effective anti-inflammatory drugs has been studied for many years [33-35].

23

The synthesis derivated in the isoandrographolide and 12-hydroxy-14-

dehydroandrographolide, and was evaluated as inhibitory activity of IL-6 and TNF-α

expression in mouse macrophages. The andrographolide derivative presented cytokines

inhibitory effect, being better than the andrographolide. On the other hand, the compound

with 12-hydroxy-14-dehydroandrographolide structure, having aryl moiety C-12, showed the

best inhibitory activity [33].

Suebsasana and collaborators [34] presented the andrographolide effect on writhing

test and carrageenan-induced paw edema. The animals were treated with andrographolide

derivatives and 12-hydroxy-14-dehydroandrographolide at dose of 4 mg/kg intraperitoneally.

Andrographolide derivatives and 12-hydroxy-14-dehydroandrographolide presented better

anti-inflammatory and analgesic effects compared with the parent compound.

Although previous studies indicated that the derivatives 12-hydroxy-14

dehydroandrographolide are the most potent, Dai and collaborators [35] proposed a different

modification: introducing the group 15-alkylidene structure of andrographolides. Hence, to

investigate whether these compounds display inflammatory properties, dimethylbenzene-

induced mouse ear edema was used, as well as rat paw edema model induced by egg albumin.

Thus, it was possible to determine that 15-alkylidene structure presented anti-inflammatory

properties, probably due to the inhibition of serum iNOS activity and PGE2. In summary, the

study demonstrated that the introduction of the p-chlorobenzylidene group in the C-15

presented better anti-inflammatory effects, probably inhibiting PGE2, inhibition of iNOS

activity and the remarkable diminution of NO production.

Hispanolone

The labdane diterpenoid hispanolone was first isolated from Ballota hispanica.

Previous studies of hispanolone and the structurally related diterpene hispanolone has

24

revealed the anti-inflammatory activity and a very low former cytotoxicity [36]. Thus, the

hispanolone and galeopsin biological activities were proposed with a series of nine

hispanolone derivatives as potential anti-inflammatory agents [37].

The data presented in this study demonstrate that two labdane diterpenoids of the

series tested, (11A) and (11B), have potent anti-inflammatory activity due to the inhibition of

the NO and PGE2 production in LPS-stimulated macrophages, probably on account of the

inhibition of NOS-2 and COX-2 expression. These effects are mediated by the inhibition of

IKK activity, which results in stabilization of the NF-κB /IκB complex and inhibition of the

NF-κB nuclear translocation.

This study corroborates with potential anti-inflammatory actions of semisynthetic

labdane derivatives and the mechanisms involved. Only studies demonstrating biological

activities by the hispanolone have been identified [38].

Ent-kaurene

Ent-kaurene diterpenes are known to have interesting biological properties, some of

these compounds have been found to be cytotoxic against several cancer cell lines [39]. Thus,

we proposed the development of potential anti-inflammatory agents for the preparation and

evaluation of anti-inflammatory activity of kaurene derivatives [40].

Hueso-Falcón and collaborators [40] synthesized 63 derivatives. Some derivatives had

no effect, however, other demonstrated consistent cytotoxicity by MTT assay. Only three of

these analog compounds, (12A), (12B) and (12C), showed the most potent anti-inflammatory

effect. The existence of a carboxylic acid seems to play an important role for NO inhibitory

activity and cell survival, since it is present in the three mentioned active compounds [40].

Therefore, the activity of these compounds may be at least in part due to its NF-kB

inhibitory activity. In addition to the inhibitory effects on NO production, these compounds

25

were able to inhibit several cytokines involved in the inflammatory response after LPS

stimulation, such as IL-6, IL-1b, TNF-α and IFN-γ.

Pseudopterosin

The pseudopterosins (PsA) is a diterpene glycoside class isolated from the marine

octocoral Pseudopterogorgia elisabethae [41]. They are quite simple molecules structurally,

consisting of a tricyclic hydrocarbon core possessing four stereocenters, and a sugar, that is

appended directly to one of the rings [42].

Zhong and collaborators [43] proposed the insertion of a methyl group between C-

glycoside and the PsA, assessing their anti-inflammatory effect from the phorbol myristate

acetate (PMA) induced inflammation in mouse ears. This paper demonstrated that this new

molecule inhibits phorbol myristate acetate (PMA) induced inflammation in mouse ears in a

dose-dependent manner, despite it was not significantly greater than the PsA. Furthermore,

the C-glycoside is an effective binding agent toward adenosine receptors A2A and A3.

Flachsmann and collaborators [44] reported the synthesis and in vivo anti-

inflammatory activity of a pseudopterosin analogues series. These ones were tested for their

ability to reduce PMA-induced mouse ear edema.

Structural modifications included the substitution degree of the hexahydrophenalene

core, different relative carbon configurations as well as variations of the sugar moiety, and the

site of glycosidation was performed. All compounds, except for one, proved to be active in

the mouse-ear assay and not professionally potency statistically differences could be

identified among the compounds. This compound presented of ketone which may contribute

to their ineffectiveness [44].

Acanthoic acid

26

Acanthoic acid is a novel pimarane-type diterpene that was first isolated from the

Acanthopanax koreanum Nakai (Araliaceae) root bark. Studies revealed that acanthoic acid

suppresses the production of IL-1β and TNF-α, being orally active and having no significant

toxicity in a rodent model of chronic inflammation [45].

Inspired by the medicinal potential of acanthoic acid, researchers sought to develop

structural modifications aiming to improve beyond the biological effects of the parent

molecule.

Lam and collaborators [46] synthesized acanthoic acid analogues and evaluated these

compounds as a TNF-α modulators. These analogues differ from acanthoic acid in the

conformation or composition of the rigid tricyclic core. Between the synthesized analogs, the

compound (15A), which features connection of methyl ester with the C-4 inhibited up to 99%

of TNF-α production, inhibition of IL-1β and IL-6 at concentrations in which it was not

cytotoxic corroborating studies. Suh and collaborators [45] revealed that C-4 modification

provides the enhanced in vitro activities.

Another study reports syntheses of acanthoic acid analog and their in vivo activities as

anti-inflammatory agents, according to Suh and collaborators [47]. The changes proposed in

this paper occurred at C-4. Suh and collaborators [45], as well as and Lam and collaborators

[46], confirmed that these changes enhance the anti-inflammatory effect.

It is demonstrated that some analogs exhibited good inhibitory activities in in vitro

assays and in NO and COX inhibition, showing that the in vivo effect was compound (15B).

Thus, the acanthoic acid analogs exhibited anti-inflammatory effects by regulation

mechanisms of pro-inflammatory cytokine and transcription factors as well as iNOS

inhibition [47].

As previously shown, the length of the linker between C-4 and the terminal carboxyl

group plays an important role for the anti-inflammatory effects of the acanthoic acid analogs.

27

Lee and collaborators [48] described the synthesis of the C-4-chain modified acanthoic acid

analogs as well as the evaluation of their inhibitory activities in NO generation in Raw 264.7

cells.

The C-4-chain length plays an important role for the NO inhibitory activity of the

acanthoic acid analogs. The C-4 extension, in the two carbon homologations, improved the

activity when the compound (15C) exhibited the most potent activity. That also suggests that

the presence of double bond in the C-4-chain is beneficial to improve NO inhibitory activity

[48].

Quinopimaric acid

Quinopimaric acid is derived from the levopimaric acids. These are abietane

diterpenoids, an important class of natural products which have been used as enantiomerically

pure starting materials for the production of highly effective drugs [49].

With this objective, the quinopimaric acid synthetic transformations and the evaluation

of their anti-inflammatory activity from carrageenan were reported [49]. In this study, it was

demonstrated that quinopimaric acid derivatives (16A), (16B), (16C), (16D) and (16E)

possessed higher anti-inflammatory activities than did diclofenac. Corroborating with this, it

was showed that quinopimaric and 3’-chloroquinopimaric acid possess anti-inflammatory

activity [50].

Triterpenes

Esculentoside

Esculentoside A (EsA) is a kind of triterpene saponin isolated from the Phytolacca

esculenta root. Studies reported that EsA inhibits inflammatory mediators secretion such as

tumor necrosis factor (TNF), interleukins (IL-1β and IL-6) and prostaglandin E2 in several

28

cell types [51]. However, haemolytic activity is the main toxicity of EsA, which needs to be

overcome.

Aiming to optimize the EsA structure and explore its structure-activity relationship in

order to seek the derivatives with increased biological activity and lower toxicity, so, the EsA

structural modifications was proposed seeking to improve anti-inflammatory profile and

reduces haemolytic effect [52].

The conversion of the C-28 carboxylic acid into an amide affected its inhibitory

activity towards COX-2 and haemolytic activity. Since the most active compound was the

derivative (17A), that corroborates with the study which related the EsA derivatives showed

higher inhibitory effects on LPS-induced NO production and lower haemolytic activities than

EsA [53].

Glycyrrhizin

Glycyrrhizin (GL) is a triterpene glycoside extracted from the Glycyrrhiza glabra root,

consisting of glycyrrhetic acid (GA), a pentacyclic triterpene and two molecules of glucuronic

acid at the C-3 position [54]. The chemical modification of glycyrrhetinic enhances the

biological activity of this terpene [54]. Thus, Matsui and collaborators [55] investigated the

structure-activity relation of GL derivatives about the inhibitory effect of the chemokine

production on IL-8 and eotaxin 1.

Structural changes occurred at C-11, C-18 and C-30 positions of GL. Notably, GL-

modified compounds, homo-30-OH-GL (18A) and hetero-30-OHGL (18B) are presumed to

be good with their inhibitory activity against both IL-8 and eotaxin 1 production.

Corroborating the results, Baltina and collaborators [54] demonstrated that changes of this

kind enhance the biological effect of GL, in addition to alcoholic triterpenoids, which are in

general more active than acidic ones [56].

29

Dammarane-type

A new novel triterpene naturally occurring compound based on the dammarane

skeleton is (17α)-23-(E)-dammara-20,23-diene-3β,25-diol, which has been elucidated in the

Palmyrah palm (Borassus flabellifer) [57]. It presents very promising immunosuppressive

profile in vitro and in vivo. Based on the promising biological properties, we investigated the

influence of the configuration of the (C-17) substituent about the anti-inflammatory effect

[58].

This way, we identified compounds which are more thermodynamically stable and

with a synthetically better accessible C-17β configuration, particularly (19A), which exhibits

better in vivo activity from allergic contact dermatitis

Lupane

Lupane is pentacyclic triterpenoid, biosynthetically derived from the cyclization of

squalene and a vast class of natural products. Its structural diversity includes a wide array of

functional groups, including the betulin and its betulinic and betulonic acids derivatives [59].

Targeting the investigation of the biological activities of lupane, Reyes and

collaborators [60] isolated 19 lupane triterpenes from the Maytenus cuzcoina root and bark,

and the Maytenus chiapensis leaves, synthesizing the betulin analogues and rigidenol from the

acetylation reaction. Then, they investigated their pharmacological activity as inhibitors of

NO and PGE2 production in macrophages.

Reyes and collaborators [60] concluded that the acetylation of betulin C-28 increases the

potency and reduces the cytotoxicity of this compound. Also, the acetylation of rigidenol at

C-11 (20A) or the chlorination at C-30 (20B) increases the potency of the compound. That

was expected because Huguet and collaborators [56] assumed that lupane derivatives are

more active when they are present in the carboxyl groups.

30

Betulinic acid

Betulinic acid, a pentacyclic triterpene discovered in 1995, is a compound isolated

from various plants widespread in tropical regions (e.g., Tryphyllum peltatum, Ancistrocladus

heyneaus, Zizyphus joazeiro, Diospyoros leucomelas, Tetracera boliviana and Syzygium

formosanum) [61, 62]. It was reported that the combination of enone functionalities with

cyano and carboxyl groups in ring A and an enone functionality in ring C is an essential

structural feature for high potency in various bioassays related to inflammation [63].

The inhibitory activities of new synthetic triterpenoids on NO production induced by

IFN-γ in mouse macrophages were evaluated, and the compounds which have the group

dinitrile are much more potent than other derivatives. The 2-cyano-3,12-dioxooleana-1,9(11)-

dien-28-oic acid (CDDO) derivative, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-onitrile,

derivated of oleanolic acid, showed high inhibitory activity against the production of NO in

mouse macrophages, about 100 times more potent than the CDDO. Several of these

compounds presented in vivo anti-inflammatory activity, i.p. or p.o., against peritoneal

inflammation induced by thioglycollate and IFN-γ [63].

Honda and collaborators [64] showed that several new semi-synthetic betulinic acid

analogues display highly potent anti-inflammatory activity in vitro. Moreover, the compound

(22A) was highly and orally active in vivo. In the inhibition of NO production in RAW 264.7,

cells stimulated with interferon-γ and induction of the anti-inflammatory, cytoprotective

enzyme, heme oxygenase-1 in the liver (in vivo), the compounds with a cyano enone

functionality in ring A were highly active. A similar effect was showed for the CDDO,

whereas betulinic acid was inactive. The new analogue (22A), oral dosing of 2 µM, presented

significantly more potency in vivo than both betulinic acid and the oleanolic acid analogue,

CDDO [64].

31

Recently, it was demonstrated that betulinic acid (20 and 40 mg/kg) reduced the paw

edema at 3, 4 and 5 h after λ-carrageenan administration by detecting the levels of

cyclooxygenase-2 (COX-2), nitric oxide (NO), tumor necrosis factor (TNF-α), interleukin-1β

(IL-1β) and malondialdehyde (MDA) in the tissue [65], which confirms the results obtained

from betulinic acid analogues that were described previously [64].

Novel tricyclic compounds having acetylene groups at C-8a and cyano enones in rings

A and C are a novel class of potent anti-inflammatory, cytoprotective, growth-suppressive and

pro-apoptotic compound shave. Some C-8a functionalized analogues using new tricycles as

starting materials were used and evaluated as to their potency for inhibition of NO production

in RAW 264.7 cells stimulated with interferon-γ. The compounds with acetylene groups were

the most potent in vitro and in vivo bioassays as anti-inflammatory [66].

Thus, carboxyl, methoxycarbonyl, and nitrile groups at C-2 enhanced activity, while

hydroxyl, aminocarbonyl, methoxy, chloride and bromide groups decreased it. For some

analogues, triterpenoids bearing C-28 in the carboxyl group were more potent than C-28

methyl; esters, but for other similar activity or even less potent activities, were observed when

C-28 was carboxylic acid [67].

Faradiol

Faradiol is a monoester pentacyclic triterpenoid obtained from Calendula officinalis L.

flowers [68]. With the goal to improve the anti-inflammatory activity of faradiol, Neukirch

and collaborators [69] proposed a modification of the chemical groups of the monoester or the

introduction of new functional groups. Selective chemical modifications, such as changes to

the ester function at C-3 (ring A), the free OH group at C-16 (ring D) and the C=C bond in

ring. It was proved that the substitution of methyl groups at C-30 to alcohol (23A) or to

aldehyde (23B) markedly improved the anti-inflammatory potency of faradiol [69].

32

Boswellic acids

Boswellic acids (BA's) are triterpenoid pentacyclic acids isolated from Boswellia

carterii. Several studies reported that the biological activity can highlight the inhibitory

activity of 5-lipoxygenase, the key enzyme of leukotriene biosynthesis [70]. Henkel and

collaborators [71] proposed modifications to BA’s of type C-3 (OH or acetoxy group) and C-

11 (oxo moiety present or absent) position, yielding 3-O-acetyl-11-keto-β-boswellic acid

(AKBA), 3-O-acetyl-β-boswellic acid (Aβ-BA), 11-keto-β-boswellic acid (KBA) and β-

boswellic acid (β-BA). For assessing the anti-inflammatory effect of BA's derivatives, the

LPS activities and of iNOS expression were verified.

Polar residues were found in C-3 position and the absence of the 11-keto group are

structural determinants required for the inhibition of LPS activity and LPS-induced iNOS

expression inhibition without significantly affecting cell viability up to 10 µM [71]. Setting a

paradox, Siddiqui and collaborators [72] found that this dual inhibitory action on the

inflammatory process is unique to BA’s. Of these BA's derivates, 3-acetyl-11-keto-β-

boswellic acid (AKBA) is the most potent inhibitor of 5-LO, an enzyme responsible for

inflammation.

The need to find drugs which can effectively attenuate inflammation led the

researchers to search drugs derived from structural changes in terpenes. This review shows

terpenes, as betulinic acid and andrographolide that suffered some structural changes, getting

more active compounds than the parent molecule. The importance of the terpenes structural

change in the search for an effective drug led the researchers to discover docetaxel, a

derivative of taxol (diterpene) more potent against cancer cells. That leads us to believe that

the terpenes modification is an interesting tool for the discovery of a drug with a good anti-

inflammatory effect.

REFERENCES

33

1 Ferrero-Miliani L, Nielsen OH, Andersen PS, Girardin SE. Chronic inflammation:

importance of NOD2 and NALP3 in interleukin-1beta generation. Clin Exp Immunol

2007;147(2):227-35.

2 De Cassia da Silveira e Sa R, Andrade LN, De Sousa DP. A review on anti-inflammatory

activity of monoterpenes. Molecules 2013;18(1):1227-54.

3 Makhija DT, Somani RR, Chavan AV. Synthesis and Pharmacological Evaluation of

Antiinflammatory Mutual Amide Prodrugs. Indian J Pharm Sci 2013;75(3):353-7.

4 Srinivasan K, Muruganandan S, Lal J, Chandra S, Tandan SK, Prakash VR. Evaluation of

anti-inflammatory activity of Pongamia pinnata leaves in rats. J Ethnopharmacol 2001;78(2-

3):151-7.

5 Sato T. Unique biosynthesis of sesquarterpenes (C35 terpenes). Biosci Biotechnol Biochem

2013;77(6):1155-9.

6 Guimarães AG, Quintans JSS, Quintans-Júnior LJ. Monoterpenes with Analgesic Activity-

A Systematic Review. Phytother Res 2013;27:1-15.

7 Quintans JSS, Menezes PP, Santos MRV, Bonjardim LR, Almeida JRGS, Gelain DP, et al.

Improvement of p-cymene antinociceptive and anti-inflammatory effects by inclusion in β-

cyclodextrin. Phytomedicine 2013;20:436-40.

8 De Sousa DP, Quintans-Júnior LJ, Almeida RN. Evaluation of the anticonvulsant activity of

alfa-Terpineol. Pharm Biol 2007;45:69-70.

9 Silva-Filho JC, Oliveira NNPM, Arcanjo DDR, Quintans-Júnior LJ, Cavalcanti SCH,

Santos MRV, et al. Investigation of Mechanisms Involved in (-)-Borneol-Induced

Vasorelaxant Response on Rat Thoracic Aorta. Basic Clin Pharmacol Toxicol 2012;110:171-

7.

10 Gertsch J, Leonti M, Raduner S, Racz I, Chen JZ, Xie XQ, et al. Beta-caryophyllene is a

dietary cannabinoid. P Natl Acad Sci USA 2008;105(26):9099-104.

11 Quintans-Júnior LJ, Guimarães AG, Santana MT, Araújo BES, Moreira FV, Bonjardim

LR, et al. Citral reduces nociceptive and inflammatory response in rodents. Braz J

Pharmacogn 2011;21:497-502.

12 Martin S, Padilla E, Ocete MA, Galvez J, Jimenez J, Zarzuelo A. Anti-inflammatory

activity of the essential oil of Bupleurum fruticescens. Planta Med 1993;59(6):533-6.

13 Quintans-Junior L, Da Rocha RF, Caregnato FF, Moreira JC, Da Silva FA, Araujo AA, et

al. Antinociceptive action and redox properties of citronellal, an essential oil present in

lemongrass. J Med Food 2011;14(6):630-9.

14 Hirota R, Roger NN, Nakamura H, Song HS, Sawamura M, Suganuma N. Anti-

inflammatory effects of limonene from yuzu (Citrus junos Tanaka) essential oil on

eosinophils. J Food Sci 2010;75(3):H87-92.

15 Chen YC, Li YC, You BJ, Chang WT, Chao LK, Lo LC, et al. Diterpenoids with anti-

inflammatory activity from the wood of Cunninghamia konishii. Molecules 2013;18(1):682-9.

16 Newman DJ, Cragg GM. Natural products as sources of new drugs over the last 25 years. J

Nat Prod 2007;70(3):461-77.

17 Harvey AL. Natural products in drug discovery. Drug Discov Today 2008;13(19-20):894-

901.

18 Pontiki E, Hadjipavlou-Litina D, Litinas K, Nicolotti O, Carotti A. Design, synthesis and

pharmacobiological evaluation of novel acrylic acid derivatives acting as lipoxygenase and

cyclooxygenase-1 inhibitors with antioxidant and anti-inflammatory activities. Eur J Med

Chem 2011;46(1):191-200.

19 Abdou WM, Ganoub NA, Sabry E. Synthesis and quantitative structure-activity

relationship study of substituted imidazophosphor ester based tetrazolo[1,5-b]pyridazines as

antinociceptive/anti-inflammatory agents. Beilstein J Org Chem 2013;9:1730-6.

34

20 Porto TS, Furtado NA, Heleno VC, Martins CH, Da Costa FB, Severiano ME, et al.

Antimicrobial ent-pimarane diterpenes from Viguiera arenaria against Gram-positive

bacteria. Fitoterapia 2009;80(7):432-6.

21 Park YD, Jin CH, Choi DS, Byun MW, Jeong IY. Biological evaluation of

isoegomaketone isolated from Perilla frutescens and its synthetic derivatives as anti-

inflammatory agents. Arch Pharm Res 2011;34(8):1277-82.

22 Chib R, Shah BA, Anand N, Pandey A, Kapoor K, Bani S, et al. Psilostachyin, acetylated

pseudoguaianolides and their analogues: preparation and evaluation of their anti-inflammatory

potential. Bioorg Med Chem Lett 2011;21(16):4847-51.

23 De Las Heras B, Rodriguez B, Bosca L, Villar AM. Terpenoids: sources, structure

elucidation and therapeutic potential in inflammation. Curr Top Med Chem 2003;3(2):171-85.

24 Neukirch H, Kaneider NC, Wiedermann CJ, Guerriero A, D'Ambrosio M. Parthenolide

and its photochemically synthesized 1(10)Z isomer: chemical reactivity and structure-activity

relationship studies in human leucocyte chemotaxis. Bioorgan Med Chem 2003;11(7):1503-

10.

25 Kwok BH, Koh B, Ndubuisi MI, Elofsson M, Crews CM. The anti-inflammatory natural

product parthenolide from the medicinal herb Feverfew directly binds to and inhibits IkappaB

kinase. Chem Biol 2001;8(8):759-66.

26 Zhang Y, Li Y, Guo YW, Jiang HL, Shen X. A sesquiterpene quinone, dysidine, from the

sponge Dysidea villosa, activates the insulin pathway through inhibition of PTPases. Acta

Pharm Sinic 2009;30(3):333-45.

27 Petronzi C, Filosa R, Peduto A, Monti MC, Margarucci L, Massa A, et al. Structure-based

design, synthesis and preliminary anti-inflammatory activity of bolinaquinone analogues. Eur

J Med Chem 2011;46(2):488-96.

28 Amigo M, Terencio MC, Mitova M, Iodice C, Paya M, De Rosa S. Potential antipsoriatic

avarol derivatives as antioxidants and inhibitors of PGE(2) generation and proliferation in the

HaCaT cell line. J Nat Prod 2004;67(9):1459-63.

29 Amigo M, Paya M, De Rosa S, Terencio MC. Antipsoriatic effects of avarol-3'-

thiosalicylate are mediated by inhibition of TNF-alpha generation and NF-kappa B activation

in mouse skin. Brit J Pharmacol 2007;152(3):353-65.

30 Laube T, Bernet A, Dahse HM, Jacobsen ID, Seifert K. Synthesis and pharmacological

activities of some sesquiterpene quinones and hydroquinones. Bioorgan Med Chem

2009;17(4):1422-7.

31 Dufort I, Labrie F, Luu-The V. Human types 1 and 3 3 alpha-hydroxysteroid

dehydrogenases: differential lability and tissue distribution. J Clin Endocrinol Metab

2001;86(2):841-6.

32 Grozio A, Millo E, Guida L, Vigliarolo T, Bellotti M, Salis A, et al. Functional

characterization of a synthetic abscisic acid analog with anti-inflammatory activity on human

granulocytes and monocytes. Biochem Bioph Res Co 2011;415(4):696-701.

33 Li J, Huang W, Zhang H, Wang X, Zho, H. Synthesis of andrographolide derivatives and

their TNF-alpha and IL-6 expression inhibitory activities. Bioorg Med Chem Lett

2007;17(24):6891-4.

34 Suebsasana S, Pongnaratorn P, Sattayasai J, Arkaravichien T, Tiamkao S, Aromdee C.

Analgesic, a ntipyretic, anti-inflammatory and toxic effects of andrographolide derivatives in

experimental animals. Arch Pharm Res 2009:32(9):1191-200.

35 Dai GF, Zhao J, Jiang ZW, Zhu LP, Xu HW, Ma WY, et al. Anti-inflammatory effect of

novel andrographolide derivatives through inhibition of NO and PGE2 production. Int

Immunopharmacol 2011;11(12):2144-9.

35

36 Nieto-Mendoza E, Guevara-Salazar JA, Ramirez-Apan MT, Frontana-Uribe BA, Cogordan

JA, Cardenas J. Electro-oxidation of hispanolone and anti-inflammatory properties of the

obtained derivatives. J Org Chem 2005;70(11):4538-41.

37 Giron N, Traves PG, Rodriguez B, Lopez-Fontal R, Bosca L, Hortelano S, De Las Heras

B. Suppression of inflammatory responses by labdane-type diterpenoids. Toxicol Appl Pharm

2008;228(2):179-89.

38 Chinou I. Labdanes of natural origin-biological activities (1981-2004). Curr Med Chem

2005;12(11):1295-317.

39 Ghisalberti E. The biological activity of naturally occurring kaurane diterpenes. Elsevier:

Amsterdam, PAYS-BAS, 1997;68:303-25.

40 Hueso-Falcon I, Cuadrado I, Cidre F, Amaro-Luis JM, Ravelo AG, Estevez-Braun A, et al.

Synthesis and anti-inflammatory activity of ent-kaurene derivatives. Eur J Med Chem

2011;46(4):1291-305.

41 Roussis V, Wu Z, Fenical W, Strobel SA, Van Duyne GD, Clardy J. New anti-

inflammatory pseudopterosins from the marine octocoral Pseudopterogorgia elisabethae. J

Org Chem 1990;55(16):4916-22.

42 Zhong W, Little RD. Exploration and determination of the redox properties of the

pseudopterosin class of marine natural products. Tetrahedron 2009;65(52):10784-90.

43 Zhong W, Moya C, Jacobs RS, Little RD. Synthesis and an evaluation of the bioactivity of

the C-glycoside of pseudopterosin A methyl ether. J Org Chem 2008;73(18):7011-6.

44 Flachsmann F, Schellhaas K, Moya CE, Jacobs RS, Fenical W. Synthetic pseudopterosin

analogues: A novel class of antiinflammatory drug candidates. Bioorgan Med Chem

2010;18(23): 8324-33.

45 Suh YG, Kim YH, Park MH, Choi YH, Lee HK, Moon JY, et al. Pimarane

cyclooxygenase 2 (COX-2) inhibitor and its structure-activity relationship. Bioorg Med Chem

Lett 2001;11(4):559-62.

46 Lam T, Ling T, Chowdhury C, Chao TH, Bahjat FR, Lloyd GK, et al. Synthesis of a novel

family of diterpenes and their evaluation as anti-inflammatory agents. Bioorg Med Chem Lett

2003; 13(19):3217-21.

47 Suh YG, Lee KO, Moon SH, Seo SY, Lee YS, Kim SH, et al. Synthesis and anti-

inflammatory effects of novel pimarane diterpenoid analogs. . Bioorg Med Chem Lett

2004;14(13):3487-90.

48 Lee KO, Min KH, Suh YG. Synthesis of C4-modified acanthoic acid analogs and their

biological evaluation as nitric oxide inhibitors. Arch Pharm Res 2005;28(6):648-51.

49 Kazakova OB, Tret'iakova EV, Smirnova IE, Spirikhin LV, Tolstikov GA, Chudov EV,

Synthesis and anti-inflammatory activity of quinopimaric acid derivatives. Bioorg Khim

2010;36(2):277-82.

50 Flekhter OB, Smirnova IE, Tret'iakova EV, Tolstikov GA, Savinova OV, Boreko EI.

Synthesis of dihydroquinopymaric acid conjugates with amino acids. Bioorg Khim

2009;35(3):424-30.

51 Xiao ZY, Zheng QY, Jiang YY, Zhou B, Yin M, Wang HB, et al. Effects of esculentoside

A on production of interleukin-1, 2, and prostaglandin E2. Acta Pharm Sinic 2004;25:6:817-

21.

52 Wu F, Yi Y, Sun P, Zhang, D. Synthesis, in vitro inhibitory activity towards COX-2 and

haemolytic activity of derivatives of esculentoside A. Bioorg Med Chem Lett

2007;17(23):6430-3.

53 Gong W, Jiang Z, Sun P, Li L, Jin Y, Shao L, et al. Synthesis of novel derivatives of

esculentoside A and its aglycone phytolaccagenin, and evaluation of their haemolytic activity

and inhibition of lipopolysaccharide-induced nitric oxide production. Chem Biodivers

2011;8(10):1833-52.

36

54 Baltina LA. Chemical modification of glycyrrhizic acid as a route to new bioactive

compounds for medicine. Curr Med Chem 2003;10(2):155-71.

55 Matsui S, Matsumoto H, Sonoda Y, Ando K, Aizu-Yokota E, Sato T, et al. Glycyrrhizin

and related compounds down-regulate production of inflammatory chemokines IL-8 and

eotaxin 1 in a human lung fibroblast cell line. Int Immunopharmacol 2004;4(13):1633-44.

56 Huguet A, Del Carmen Recio M, Manez S, Giner R, Rios J. Effect of triterpenoids on the

inflammation induced by protein kinase C activators, neuronally acting irritants and other

agents. Eur J Pharmacol 2000;410(1):69-81.

57 Revesz L, Hiestand P, La Vecchia L, Naef R, Naegeli HU, Oberer L, et al. Isolation and

synthesis of a novel immunosuppressive 17alpha-substituted dammarane from the flour of the

Palmyrah palm (Borassus flabellifer). Bioorg Med Chem Lett 1999;9(11):1521-6.

58 Scholz D, Baumann K, Grassberger M, Wolff-Winiski B, Rihs G, Walter H, et al.

Synthesis of dammarane-type triterpenoids with anti-inflammatory activity in vivo. Bioorg

Med Chem Lett 2004;14(11):2983-6.

59 Grishko VV, Tolmacheva IA, Galaiko NV, Pereslavceva AV, Anikina LV, Volkova LV, et

al. Synthesis, transformation and biological evaluation of 2,3-secotriterpene acetylhydrazones

and their derivatives. Eur J Med Chem 2013;68C:203-211.

60 Reyes CP, Nunez MJ, Jimenez IA, Busserolles J, Alcaraz MJ, Bazzocchi IL. Activity of

lupane triterpenoids from Maytenus species as inhibitors of nitric oxide and prostaglandin E2.

Bioorg Med Chem 2006;14(5):1573-9.

61 Pisha E, Chai H, Lee IS, Chagwedera TE, Farnsworth NR, Cordell GA, et al., Discovery of

betulinic acid as a selective inhibitor of human melanoma that functions by induction of

apoptosis. Nat Med 1995;1(10):1046-51.

62 Selzer E, Pimentel E, Wacheck V, Schlegel W, Pehamberger H, Jansen B, et al. Effects of

betulinic acid alone and in combination with irradiation in human melanoma cells. J Invest

Dermatol 2000;114(5):935-40.

63 Honda T, Honda Y, Favaloro FGJr, Gribble GW, Suh N, Place AE, et al. A novel

dicyanotriterpenoid, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-onitrile, active at picomolar

concentrations for inhibition of nitric oxide production. Bioorg Med Chem Lett

2002;12(7):1027-30.

64 Honda T, Liby KT, Su X, Sundararajan C, Honda Y, Suh N, et al. Design, synthesis, and

anti-inflammatory activity both in vitro and in vivo of new betulinic acid analogues having an

enone functionality in ring A. Bioorg Med Chem Lett 2006;16(24):6306-9.

65 Tsai JC, Peng WH, Chiu TH, Lai SC, Lee CY. Anti-inflammatory effects of Scoparia

dulcis L. and betulinic acid. Am J Chin Med 2011;39(5):943-56.

66 Honda T, Sundararajan C, Yoshizawa H, Su X, Honda Y, Liby KT, et al. Novel tricyclic

compounds having acetylene groups at C-8a and cyano enones in rings A and C: highly

potent anti-inflammatory and cytoprotective agents. J Med Chem 2007;50(8):1731-4.

67 Sultana N, Saify ZS. Naturally occurring and synthetic agents as potential anti-

inflammatory and immunomodulants. Antiinflamm Antiallergy Agents Med Chem

2012;11(1):3-19.

68 Zitterl-Eglseer K, Sosa S, Jurenitsch J, Schubert-Zsilavecz M, Della Loggia R, Tubaro A,

et al. Anti-oedematous activities of the main triterpendiol esters of marigold (Calendula

officinalis L.). J Ethnopharmacol 1997;57(2):139-44.

69 Neukirch H, D'Ambrosio M, Sosa S, Altinier G, Della Loggia R, Guerriero A. Improved

anti-inflammatory activity of three new terpenoids derived, by systematic chemical

modifications, from the abundant triterpenes of the flowery plant Calendula officinalis. Chem

Biodivers 2005;2(5):657-71.

70 Safayhi H, Rall B, Sailer ER, Ammon HP. Inhibition by boswellic acids of human

leukocyte elastase. J Pharmacol Exp Ther 1997;281(1):460-3.

37

71 Henkel A, Kather N, Monch B, Northoff H, Jauch J, Werz O. Boswellic acids from

frankincense inhibit lipopolysaccharide functionality through direct molecular interference.

Biochem Pharmacol 2012;83(1):115-21.

72 Siddiqui MZ. Boswellia serrata, a potential antiinflammatory agent: an overview. Indian J

Pharm Sci 2011;73(3):255-61.

38

Figure 1. Flowchart of included studies.

755

Figure 2: Structures of terpenes derivatives.

O

O

O

O

(1) (1A)

O O

O

HO

O O

OO

NN

HO

(2) (2A)

O OO

O

HON

N

(2B)

Scopus (n=13) Embase (n=304) Pubmed (n= 445)

Articles that did not meet inclusion

criteria, based in titles, abstract or

full text

27 articles selected

Exclusion of repetitions

39

O OO

O

HO

(2C)

OO

O O O

H

OH

OMe

(3) (3A)

O

H

OMe

OHO

(3B)

OOH O

(3C)

O

O

H

HO

OMe

O

O

H

HO

(4) (4A)

40

H

OH

HO

H

OH

HO

S

HOOC

(5) (5A)

H

OH

OH

CHO

NaO3SO

O

O OMe

OMe

(6) (6A)

O

O

O

H

(6B)

O CH3

OH

OHO O CH3

OHO

OH

O

(7) (7A)

41

HO

H

O

O

HO

HO

HO

H

O

O

O C

O

R

(8) (8A) R= C6H5

(8B) R= C6H5NO2

(8C) R= C6H5CH3

OHH

OH

O

O

OH

OHH

OH

O

O

OiPr

(9) (9A)

OHH

O C (CH2)14CH3

O

O

O

(9B)

42

OHH

OH

O

O

OH

OH

O

O

OC

O

NO

Cl

(10) (10A)

H

OH

O

O

H

O

O

(11) (11A)

H

O

O

O

HO

(11B)

43

COOHH

H

COOH

O

H

(12) (12A)

COOCH2TMS

H OH

O

(12B)

COOCH2PhH

H OH

O

(12C)

H

OH

O

O

OH

OH

OH

H

OMe

CH2

O

OH

OH

OH

(13) (13A)

H

OH

O

O

OH

OH

OH

OG

OH

H

(14) (14A)

44

OG

OHH

O

(14B)

COOH C O

O

CH3

(15) (15A)

COOH

(15B)

OH (15C)

COOH

O

O

COOCH3

OH

O

(16) (16A)

45

COOCH3

OH

OH

(16B)

COOCH3

OH

N OH

(16C)

COOCH3

OAc

N OAc

(16D)

COOCH3

NO

HO

H

(16E)

46

O

HOCOOH

GOH

O

O

G

O

HO

GOH

O

O

G

NH

O

O

O

(17) (17A)

O

COOH

GG

H

OH

O

CH2OH

GG

H

(18) (18A)

O

CH2OH

GG

H

(18B)

HO

H

H

OH

HO

H

H

H

H

NH2

(19) (19A)

47

HO

HO

AcO

O

(20) (20A)

AcO

H2C

O

Cl

(20B)

COOH

OH

CN

O

CN

OH

CN

O

(21) (21A)

OH

CN

O

CN

(21B)

48

COOH

H

O

HO

H

H

H

O

O

H

HCN

H

N

O

N

(22) (22A)

HHO

H

H

H

H

OH

HO

CHO

OH

(23) (23A)

HO

CH2OH

OH

(23B)

HOOC

HO

HOOC

R

(24)

(24A) R= O

O

49

(24B) R=

O

O

OH

O

(24C) R=

HOO

O

O

(24D) R= HO O

O O

(25E) R=

HOO

O

50

Table 1. Description of the modification chemical of the terpenes and pharmacological aspects of the studies included in systematic review.

Ref Terpene Source Methods used DE/CE Route Animal/Cell Result Country

Park et al.,

2011

Isoegomaketone (1) Isolated from

Perilla

frutescens

Me NO; Me MCP-1;

Me IL-6; LA NF-kB;

LA AP-1

RAW 264.7 cells (1A) Korea

Chib et al.,

2011

Parthenin (2) Isolated from

Parthenium

hysterophorus

Me TNF-α; Me IL-1β;

Me IL-6

1 µg/ml

Ne Murine (2A) (2B) (2C) India

Neukirch et

al., 2003

Parthenolide (3)

Me IL-8 10 pg/ml

1 ng/ml

100 ng/ml

10 µg/ml

Ne Human (3A) (3B) (3C) Italy

Petronzi et

al., 2010

Bolinaquinone (4)

Me PGE2

RAW 264.7 cells (4A) Italy

Amigó et

al., 2004

Avarol (5)

Isolated from

Dysidea avara

Me PGE2 5 µM CHK HaCaT cell

line

(5A)

Spain

Laube et

al., 2009

Siphonodictyal (6) Synthesis Ac 3α-HSD; Pr ROS Gr (6A) (6B) Germany

Grozio et

al., 2011

Abscisic acid (7) Me PGE2; Me MCP-1;

Pr ROS; CBHG

0.5 nM;

1 nM;

10 nM;

100 nM;

1 µM;

5 nM

Gr and Mo

Human

(7A) Italy

51

Table 1 (Continued)

Ref Terpene Obtainment Methods used DE/CE Route Animal/Cell Result Country

Li et al.,

2007

Andrographolide

(8)

Me TNF-α; Me IL-6 20 µM J774A.1 cells (8A) (8B) (8C) China

Suebsasana

et al., 2009

Andrographolide

(9)

Isolated from

A. paniculata

EPICg; WT 4 mg/kg i.p. Mice and Rats

SD

(9A) (9B) Thailand

Dai et al.,

2011

Andrographolide

(10)

Furen

Medicines

Group,

Pharmaceutical

Co., Ltd.

EEIDd; EPIEA; Pr NO;

Ac iNOS

0.45

mmol/kg

0.9 mmol/kg

1.35

mmol/kg

i.g. Mice Kunming

and Rats SD

(10A) China

Girón et al.,

2008

Hispanolone (11)

Isolated from

Ballota

hispanica

Sy NO; In NOS-2; In

COX-2; Ex IL-6; Ex

mRNA; Me TNF-α;

EEITPA; Ac NF-κB;

Ac MAPK; Ac IKK

1 µM

10 µM

20 µM

50 µM

0.25 mg/ear

0.5 mg/ear

1 mg/ear

a.t. RAW 264.7 cell

and Swiss mice

(11A) (11B)

Spain

Hueso-

Falcón et

al., 2011

Ent-kaurene (12)

Synthesis Pr NO; Ex NOS-2; Ex

mRNA; Ac NF-kB; Me

IL-6

1 µM

5 µM

10 µM

25 µM

50 µM

RAW 264.7 cell (12A) (12B) (12C)

Spain

Zhong et

al., 2008

Pseudopterosin (13) EEIPMA; BAR A2A

and A3

17 μg/ear a.t. Mice (13A) USA

52

Table 1 (Continued)

Ref Terpene Obtainment Methods used DE/CE Route Animal/Cell Result Country

Flachsmann

et al., 2010

Pseudopterosin (14)

EEIPMA 25 μg/ear a.t. Mice (14A) (14B) USA

Lam et al.,

2003

Acanthoic acid (15)

Isolated

from

Perilla

frutescens

Me TNF-α HPBMC cells (15A)

USA

Suh et al.,

2004

Acanthoic acid (15)

Isolated

from

Perilla

frutescens

In COX-2; In NO;

AICFA

5 mg/kg

15 mg/kg

25 mg/kg

i.p. Raw 264.7 cells

and Rats

(15B) South

Korea

Lee et al.,

2005

Acanthoic acid (15)

Me NO Raw 264.7 cells (15C) Korea

Kazakova

et al., 2010

Quinopimaric Acid

(16)

EPICg 50 mg/kg

100 mg/kg

i.g. Rats (16A) (16B) (16C)

(16D) (16E)

Russia

Wu et al.,

2007

Esculentoside (17)

Ex hCOX-2 10 µM sf-9 cells (17A) China

Matsui et

al., 2004

Glycyrrhizin (18)

Isolated from

Glycyrrhiza

uralensis

Me IL-8; Me eotaxin 1;

Ex IL-8; Ex eotoxin 1

Ex mRNA

1 µg/ml

10 µg/ml

30 µg/ml

100 µg/ml

HFL-1 cells (18A) (18B) Japan

Scholz et

al., 2004

Dammarane-type

(19)

Syntesis ACD 0.1 M a.t. Mice (19A) Austria

53

Table 1 (Continued)

Ref Terpene Obtainment Methods used DE/CE Route Animal/Cell Result Country

Reyes et

al., 2006

Lupane (20)

Isolated from

Maytenus

cuzcoina

Pr NO; Pr PGE2 5 µM

10 µM

RAW 264.7 cell (20A) (20B) Spain

Honda et

al., 2002

CDDO (21)

Pr NO Ma (21A) USA

Honda et

al., 2007

CDDO (21)

Pr NO RAW 264.7 cells (21B)

USA

Honda et

al., 2006

Betulinic acid (22)

Pr NO RAW 264.7 cells (22A) USA

Neukirch et

al., 2005

Faradiol (23)

EEIC a.t. Mice (23A) (23B)

Italy

Henkel et

al., 2012

Boswellic acids

(24)

Isolated from

Boswellia sp

Ex iNOS 10 µM RAW 264.7 cells (24A) (24B) (24C)

(24D) (24E)

Germany

Methods abbreviations: ME, Measurement; LA, Luciferase assay; Ac, Activity; Pr, Production; CBHG, Competition Binding on Human Granulocytes;

EPICg, Carrageenan Induced Paw Edema; WT, Writhing Test; EEIDd, Dimethylbenzene Induced Ear Edema; EPIEA, Egg Albumin Induced Paw Edema;

Sy, Synthesis; In, Induction, Ex, expression; EEITPA, Tetradecanoylphorbol-13-Acetate Induced Ear edema; EEIPMA, Phorbol Myristate Acetate Induced

Ear Edema, BAR, Bind to Adenosine Receptors, AICFA, Arthritis Induced by Freund's Complete Adjuvant; ACD, Allergic Contact Dermatitis; EEIC,

Croton oil Induced Ear Edema; MCP-1, Monocyte Chemoattractant Protein 1; ROS, reactive oxygen species; 3α-HSD, 3α-Hydroxysteroid Dehydrogenase.

Abbreviations of administration routes: a.t., Administration Topically; i.g., Intragastrically; i.p., Intraperitoneally.

Abbreviations of animal/cell: Ne, Neutrophils, CHK, Cultured Human Keratinocyte; Gr, Granulocytes; Mo, Monocytes; MA, Macrophages; SD, Sprague

Dawley; HPBMC cells, Human Peripheral Blood Mononuclear; HFL-1 cells, Human Fetal Lung Fibroblastos; Sf, Spodoptera frugiperda.

54

3.2 CAPÍTULO 2

SYNTHESIS AND PHARMACOLOGICAL EVALUATION

OF CARVACROL PROPIONATE

Artigo publicado ao periódico:

Inflammation

Fator de impacto no Journal Citation Reports® (JCR):

2.457

55

Synthesis and pharmacological evaluation of carvacrol propionate

Marilia Trindade de Santana1

, Viviane Barros Silva2, Renan Guedes de Brito

1, Priscila

Laíse dos Santos3, Sócrates Cabral de Holanda Cavalcanti

2, Emiliano Oliveira

Barreto4, Jamylle Nunes de Souza Ferro

4, Márcio Roberto Viana dos Santos

2, Adriano

Antunes de Sousa Araújo2, Lucindo José Quintans-Júnior

1,*

1Department of Physiology. Federal University of Sergipe, São Cristovão, Brazil.

2Department of Pharmacy. Federal University of Sergipe, São Cristovão, Brazil.

3Department of Morphology, Federal University of Sergipe, São Cristovão, Brazil.

4Laboratory of Cell Biology, Federal University of Alagoas, Maceió, Brazil.

*Corresponding author: Departamento de Fisiologia, Universidade Federal de Sergipe-

UFS, Av. Marechal Rondom, s/n, São Cristóvão, Sergipe-Brazil. Tel.: +55-79-

21056645; fax: +55-79-3212-6640. E-mail address: lucindojr@gmail.com;

lucindo@pq.cnpq.br

56

Abstract

This study aimed at synthesizing the carvacrol propionate (CP) and evaluating its

pharmacological profile. CP was obtained from carvacrol and propionyl chloride

through an esterification reaction. Male Swiss mice were treated with CP (25, 50 or 100

mg/kg). We evaluated the analgesic effect, mechanical hyperalgesia and anti-

inflammatory effect. Pretreatment with CP inhibited (p < 0.01 and 0.001) the formalin-

induced nociception in both phases. CP inhibited (p < 0.05, 0.01 and 0.001) the

development of mechanical hyperalgesia. CP was able to decrease the leukocyte

recruitment (p < 0.001) and the amount of TNF-α (p < 0.001), IL-1β (p < 0.05) and

protein leakage (p < 0.01) into the pleural cavity. In addition, the paw edema was

inhibited by CP (p < 0.05, 0.01 and 0.001). The CP attenuates nociception, mechanical

hyperalgesia and inflammation, through an inhibition of cytokines.

Key-words: Terpene, carvacrol propionate, hyperalgesia, inflammation, pain.

57

1.0 Introduction

The inflammatory response is an important cause of painful conditions, resulting

from tissue injury. The tissue damage occurs due to the accumulation of various cell

types, such as masts, basophils, platelets, macrophages, neutrophils, endothelial cells,

keratinocytes and fibroblasts [1]. These cells produce a variety of mediators, such as

neurotrophic factors, neuropeptides, prostanoids and kinins which, by acting on their

own receptors, contribute to alter the firing pattern of the primary sensory neurons,

leading to inflammatory pain. Those are peripheral sensitization changes in the chemical

environment of the nerve fiber [2].

Currently, there are two approaches often used as therapeutic management for

the inflammatory pain. The first clinical alternative and the most widely used is the

Non-steroidal anti-inflammatory drugs (NSAIDs), which block the formation of pro-

inflammatory mediators, reducing the inflammatory pain by inhibiting the

cyclooxygenases (COX-1 and COX-2), as, for example, the aspirin, indomethacin and

ketoprofen [3]. The second treatment option is the desensitization of nociceptors

through the stimulation of expression of potassium channels, which hyperpolarize the

cell, decreasing the established hyperalgesia, such as opioid drugs [4].

However, new strategies for treating the inflammatory pain are needed, once the

current treatment is limited because of side effects and tolerance [5, 6]. Thus, great

effort has been expended on the development of drugs for the treatment of

inflammation.

In this context, natural products are employed worldwide in folk medicine to

treat different painful and inflammatory conditions [7, 8]. Plants, fungi, marine

organisms and bacteria are the source of potentially active chemical substances, being

considered as raw materials, i.e, the starting point for the discovery of new

58

pharmacologically active molecules [9-11]. Most drugs used in pre-clinical or clinical

studies are of natural origin and have been developed from these structural changes

[12]. The structural modification of natural products showed promising activities that

must be seen as an interesting source of new structures, with the possibility of

presenting an important biological activity [13].

Within the natural products, we can highlight the monoterpenes, main chemical

constituents of the plant essential oils with anti-inflammatory properties. Recently,

Guimarães et al. [8] suggested that monoterpenes are possible candidates for the

treatment of painful conditions. These results were corroborated by De Cassia da

Silveira e Sá et al. [7], who identified 32 monoterpenes with anti-inflammatory activity,

such as menthol, citral, (±)-citronellal, (+)-limonene, thymol, carvacrol, linalylacetate

and linalool, among others.

Aiming to improve the biological activity, the research has modified the

structure of monoterpenes, through specific chemical reactions, resulting on derivatives

[14]. Studies have shown the importance of these chemical modifications. Hydroxy-di-

hydrocarvone, which is a synthetic derivative of carvone, possesses anti-inflammatory

[15] and antinociceptive activity [16]. Carvone or active analogs inhibit nerve

excitability in accordance with different chemical structures [17]. According to De

Sousa et al. [18], monoterpenes properly derivatized enable results on new analgesic

drugs. For example, proprionate of carvacrol (CP), which is a monoterpene derivative

obtained by the esterification of carvacrol. Although its synthesis is known, there is only

one study demonstrating its antimicrobial activity [19]. Hence, it is necessary to conduct

studies to evaluate the pharmacological activity of carvacrol propionate in models of

nociception, hyperalgesia and inflammation.

59

2.0 Materials and Methods

2.1 Drugs and reagents

Carrageenan (CG), tumor necrosis factor-alpha (TNF-α), prostaglandins-E2

(PGE2), dopamine (DA), cremophor, carvacrol, propionylchloride,

ethylenediaminetetraacetic acid (EDTA), Griess reagent, Türk solution and 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from

Sigma (Saint Louis, MO, USA). Enzyme-linked immunosorbent assay (ELISA) for

mouse´s quantitative determination of TNF-α and IL-1β was obtained from BD-

Bioscience Pharmingen (San Diego, CA, USA). Indomethacin and dipyrone were

obtained from União Química (São Paulo, Brazil). Diazepam (DZP) was purchased

from Cristália (São Paulo, Brazil). Ethyl acetate, hexane and triethylamine were

obtained from Vetec (Rio de Janeiro, Brazil).

The CP was dissolved in 0.9% saline and 0.2% cremophor, used as an emulsion,

for pharmacological experiments. The other substances were solubilized with distilled

water or saline. In these protocols, the agents were injected intraperitoneally (i.p.) at

volumes of 0.1 mL/10 g. All doses and route of administration of the CP were chosen

according to Quintans-Júnior et al. [20] and Guimarães et al. [21].

2.2 Synthesis and characterization of carvacrol propionate (CP)

CP was prepared from carvacrol using the method of Dolly and Barba [22] and

characterized by 1H and

13C nuclear magnetic resonance, mass spectrometry and

infrared spectroscopy.

To obtain the CP, carvacrol (5.15 mL; 33.33 mmol) dissolved in THF was added

to propionyl chloride (4.62 mL; 50 mmol) in THF to form the ester derivative in the

60

presence of triethylamine (5.07mL; 50 mmol). The reaction was stirred for 2 h at room

temperature. The reaction mixture was concentrated under vacuum, diluted with water

and extracted with dichloromethane. The organic layer was washed with water, and

dried over Na2SO4. The solvent was distilled off and the residue purified through silica

gel column chromatography (Hex:EtOAc, 99:1) yielding CP 72.81% as a yellowish oil.

The compound obtained was characterized by 1H and

13C NMR, mass spectrometry and

infrared spectroscopy.

NMR data were recorded on a Bruker DRX400 spectrometer using CDCl3 as

solvent and tetramethylsilane (TMS) as an internal standard, and the chemical shifts are

reported in ppm (). Coupling constants (J) are reported in hertz (Hz). The abbreviations

used are s (singlet), d (doublet), t (triplet), q (quadruplet), sept (septuplet). FT-IR was

recorded on a Perkin Elmer Spectrum BX FT-IR System. Mass spectra were recorded

on a Shimadzu GCMS-QP2010S Gas Chromatograph Mass Spectrometer (equipped

with an AOC-20S auto sampler).

2.3 Animals

Adult (approximately 3 months old) male Swiss mice (28-32 g) were randomly

housed in appropriate cages at 21 2°C on a 12 h light/dark cycle (lights on 06:00 a.m.

to 6:00 p.m.), with free access to food (Purina®

, Brazil) and tap water. All experiments

were carried out between 09:00 a.m. and 16:00 p.m. in a quiet room. All nociceptive,

hyperalgesia and inflammatory tests were carried out by the same visual observer,

double-blinded and all efforts were made to minimize both the number of animals and

any discomfort inflicted upon them. Experimental protocols were approved by the

Animal Care and Use Committee at the Federal University of Sergipe (CEPA/UFS #

61

35/12) and handling procedures were in accordance with the International Council for

Laboratory Animal Science (ICLAS) and National Institute of Health (NIH).

2.4 Formalin induced nociception

The formalin test was carried out as described by Hunskaar and Hole [23]. The

animals were treated with the vehicle (saline + cremophor 0.2%), CP (25, 50, and 100

mg/kg, i.p.) or morphine (3 mg/kg, i.p.) 30 min before the formalin injection. Formalin

(1%; 20 μL) was injected into the dorsal surface of the right hind paw using a

microsyringe with a 26-gauge needle. These mice were individually placed in a

transparent plexiglass cage observation chamber (25 cm × 15 cm × 15 cm). The amount

of time spent licking the injected paw was indicative of pain. The number of lickings

from 0-5 min (first phase) and 15-30 min (second phase) was counted after the injection

of formalin.

2.5 Hot-plate test

The hot-plate test was used according to Kuraishi et al. [24]. The animals were

placed on an aluminum plate that was adapted to a water bath at 55 ± 0.5°C. The

reaction time was noted by observing the licking of the hind paws at basal, 0.5, 1.0, 1.5,

and 2.0 h after i.p. administration of vehicle, CP or morphine to different groups of 6

mice.

2.6 Hyperalgesia induced by CG, TNF-a, PGE2 and dopamine

This study was performed according to Cunha et al. [25] and Villarreal et al.

[26]. Mice were divided into five groups (n = 6, per group), which were treated with

vehicle (saline + cremophor 0.2% v/v, i.p.), CP (25, 50 or 100 mg/kg, i.p.),

62

indomethacin (10 mg/kg, i.p.) or dipyrone (60 mg/kg, i.p.). Thirty minutes after

treatment, 20 µL of CG (300 µg/paw), PGE2 (100 ng/paw), DA (30 µg/paw) or TNF-α

(100 pg/paw) were injected subcutaneously into the subplantar region of the hind paw.

The degree of hyperalgesia was evaluated at 30, 60, 120 and 180 min after the injection

of algogen agents.

2.7 Measurement of mechanical hyperalgesia

Mechanical hyperalgesia was tested in mice as reported by Cunha et al. [25]. In

a quiet room, mice were placed in acrylic cages (12 x 10 x 17 cm) with wire grid floors

for 15-30 min. before starting the test. This method consisted of evoking a hind paw

flexion reflex with a hand-held force transducer (electronic anesthesiometer; Insight®,

Ribeirão Preto, São Paulo, Brazil) adapted with a polypropylene tip. The investigator

was trained to apply the tip perpendicularly to the central area of the hind paw with a

gradual increase in pressure. The end point was characterized by the withdrawal of the

paw followed by clear flinching movements. After the paw withdrawal, the intensity of

the pressure was automatically recorded. The intensity of stimulus was obtained by

averaging four measurements taken with minimal intervals of 3 min. The animals were

tested before the treatments with vehicle, CP or control drugs, and at selected times after

the injection of the nociceptive agents. The protocol was carried out blindly, where the

researcher who performed the measures did not know which group the animal belonged

to. The results are presented as the ∆ withdrawal threshold (g), calculated by the

difference between the values obtained after the treatment and before the treatment [25].

2.8 Carrageenan-induced pleurisy

63

Pleurisy was induced by intrathoracic (i.t.) injection of CG (300 μg; 0.1 mL)

diluted in sterile saline. Control animals received the same volume of vehicle. The

animals were pretreated as described above 30 min before the injection of the

inflammatory agent. Four hours after stimulation, the animals were sacrificed in a CO2

chamber; the pleural cavities were opened and washed with 1 mL of PBS (1x)

containing EDTA (10 mM). Total leukocyte counts collected in the pleural lavage were

performed on a Neubauer chamber under an optical microscope. The samples were

diluted (40x) in Türk solution. The differential leukocyte analysis was performed under

a light microscope with immersion oil objective in cytocentrifuged smears colored with

May-Grunwald-Giemsa, on which 100 cells per slide were counted. The amounts of

TNF-α and IL-1β produced in the pleural cavity were assessed 4 h after injection of CG.

The recovered pleural lavage was centrifuged at 770 xg for 10 min. TNF-α and IL-1β

were quantified on supernatant free of cells through enzyme immunoassay (ELISA)

using matched antibody pairs from R&D Systems (Minneapolis, MN, USA;

Quantikine), according to the manufacturer’s instructions. The measurement of total

protein was held collecting the fluids recovered from the pleural cavity of the animals,

which were centrifuged for 10 min at 1.500 ×g, and the total protein content was

quantified in the supernatant, at 540 nm, using the Bradford reagent.

2.9 MTT cell viability assay

The cytotoxic effect of CP on macrophages was determined using the MTT

assay method according to Mosmann [27]. Murine peritoneal macrophages (2.5×105

cells) were treated with CP at concentrations ranging from 1.0 μg/mL to 500.0 μg/mL

and were further cultured in RPMI-1640 supplemented with 10% FBS for 24 h.

64

Thereafter, the medium was replaced with fresh RPMI containing 5 mg/mL of

MTT. After additional 4 h of incubation at 37°C, the supernatant was discharged and

DMSO solution (150 μL/well) was added to each culture plate. After 15 min of

incubation at room temperature, absorbance of solubilized MTT formazan product was

spectrophotometrically measured at 540 nm. Five individual wells were assayed per

treatment and percentage of viability was determined in relation to controls

[(absorbance of treated cells/absorbance of untreated cells) x 100].

2.10 Measurement of paw edema

The effect of CP on edema formation caused by the intraplantar injection of CG

was analyzed according to the method previously reported by Levy [28]. The animals

were divided into five groups (n = 6, per group) and treated as described above. Right

paw volume was measured by the dislocation of the water column of a plethysmometer

before (time zero) and at 1, 2, 3, 4, 5 and 6 h after subplantar injection of 40 μL of CG

(1%). Paw edema was expressed (in milliliter) as the difference between the volume of

the paw after and before CG injection. The area under the curve (AUC [0–240 min]; in

milliliter per minute) was also calculated using the trapezoidal rule.

2.11 Spontaneous locomotor activity

Mice were divided into five groups (n = 6, per group) and treated with vehicle,

CP or diazepam (1.5 mg/kg; i.p.). The spontaneous locomotor activity was assessed in a

cage activity (50×50×50 cm) at 0.5, 1, and 2 h after the treatment [29].

2.12 Evaluation of the motor activity

65

Initially, mice able to remain on the rota-rod apparatus (AVS®, Brazil) longer

than 180 sec (7 rpm) were selected 24 h before the test [30]. Then, the selected animals

were divided into five groups (n = 6, per group) and treated intraperitoneally as

described above. Each animal was tested on the rota-rod and the time (sec) that they

remained on the bar for up to 180 s was recorded after 30, 60, and 120 min of the

treatment.

2.13 Statistical analysis

Data were evaluated using GraphPad Prism Software Inc. (San Diego,

California, USA) version 5.0. Formalin, hot-plate, pleurisy and MTT tests, as well as

the evaluation of the motor through the one-way analysis of variance (ANOVA) were

followed by Tukey’s test. While mechanical hyperalgesia and edema of paw the data

obtained were evaluated by the two-way analysis of variance (ANOVA) to compare the

groups and doses at all times. If a significant interaction between the factors evaluated

(treatment and time) was detected, Bonferroni´s post-test was used. The results are

presented as mean ± SEM. In all cases, the differences were considered significant if p

< 0.05.

3.0 Results

3.1 Synthesis and characterization of propionate carvacrol (CP)

The synthesis resulted in the formation of CP (Fig. 1) yielding 72.81% as clear

oil and the characterization is in agreement with previous literature data [19].

IR (film, cm-1

) 1760 (C=O). 1H RMN ( 400 MHz, CDCl3) 7.10 (d, 1H, J = 7.8 Hz,

Ar-H), 6.98 (d, 1H, J = 7.8 Hz, Ar-H), 6.85 (s, 1H, Ar-H), 2.84 (sept, 1H, J = 6.8 Hz,

CH(CH3)2), 2.56 (q, 2H, J = 7.6 Hz, CO-CH2-CH3), 2.10 (s, 3H, Ar-CH3), 1.25 (t, 3H, J

66

= 7.5 Hz, CO-CH2-CH3), 1.21 (d, 6H, J = 6.9 Hz, CH(CH3)2). 13

C NMR (100 MHz,

CDCl3): 172.5, 149.3, 147.9, 130.8, 127.1, 124.0, 119.7, 33.6, 27.6, 23.9, 15.7, 9.2.

MS (EI) m/z [M]+ 206.

3.2 Effect of CP on formalin-induced nociception

In the test of nociception induced by formalin, CP (at all doses) and morphine

reduced significantly (p < 0.001 and p < 0.01) the licking time in the neurogenic phase

(0-5 min). In the inflammatory phase (15-30 min), treatment with CP, at all doses,

reduced significantly (p < 0.001) the licking time (Fig. 2A, B).

3.3 Effect of CP on hot-plate test

When tested in the central antinociceptive model (hot-plate model), the pre-

treatment with CP resulted in significant antinociceptive activity in doses 50 and 100

mg/kg. At 30 min after oral administration, CP doses resulted in significant activity with

p < 0.05 (100 mg/kg) and p < 0.001 (50 mg/kg). Similarly, we observed, 60 min after

the oral administration, a significant antinociceptive effect (p < 0.01 and p < 0.05) for

the doses of 50 and 100 mg/kg, respectively. After 90 and 120 min, the antinociceptive

effect was also significant, at doses of 50 and 100 mg/kg, with p < 0.001 and p < 0.05

for 90 min after treatment, and p < 0.001 for 120 min after treatment. The effect of

morphine, as expected, was significant at p < 0.001 all times observed (Table 1).

3.4 Effect of CP on the CG-induced mechanical hyperalgesia

Treatment with CP (25, 50, or 100 mg/kg; i.p.) 30 min before CG administration

exhibited a significant (p < 0.05, p < 0.01 and p < 0.001) reduction of the mechanical

67

hyperalgesia induced by CG; except for (25 mg/kg) in the time of 180 min, when

compared with animals of the control group that received only vehicle (Fig. 3A).

3.5 Effect of CP on the TNF-α induced mechanical hyperalgesia

The inhibitory effect of CP on the mechanical hyperalgesia induced by TNF-α is

shown in Figure 3B. CP (25, 50, or 100 mg/kg) reduced significantly (p < 0.05, p < 0.01

and p < 0.001) mechanical hyperalgesia induced by TNF-α, at all doses and time, except

for the lowest dose (25 mg/kg) in the time of 180 min when compared with animals of

the vehicle group (Fig. 3B).

3.6 Effects of CP on the PGE2-induced mice paw mechanical hyperalgesia

The nociception was significantly reduced (p < 0.001) by dipyrone (60 mg/kg;

ip) at all times. However, CP showed a significant reduction in the doses 25, 50 and 100

mg/kg, with p < 0.001 (Fig. 3C).

3.7 Effect of CP on the DA-induced mechanical hyperalgesia

Figure 3D shows the inhibitory effect of CP on the mechanical hyperalgesia

induced by DA. Dipyrone showed reduction in nociception at all times with p < 0.001.

CP at the time of 0.5 h showed no positive effect. However, 1 h after the treatment with

the CP, the dose of 25, 50 and 100 mg/kg showed a significant decrease (p < 0.001).

Furthermore at the time of 2 h, doses of 25, 50 and 100 mg/kg significantly reduced

mechanical hyperalgesia induced by DA when compared with animals of the vehicle

group (p < 0.001, p<0.05 and p < 0.001), respectively. However, at the last observation

time, all doses are significantly efficient (p < 0.01 and p < 0.001) in reduction of

mechanical hyperalgesia induced by DA.

68

3.8 Effect of CP on carrageenan-induced pleurisy

All doses of CP (25, 50 and 100 mg/kg) were able to suppress significantly (p <

0.001) the recruitment of leukocytes to the mouse´s pleural cavity; similar results were

obtained with the positive control, indomethacin, as shown in Fig. 4A. Pretreatment

with CP significantly reduced (p < 0.001 and p < 0.05), in all doses, the migration of

neutrophils, as shown in Fig. 4B. This inhibition is not related to cytotoxicity, since the

CP, at concentrations of 1, 10, 100 and 250 μg/mL, did not change the morphological

profile of polymorphonuclear cells in the MTT protocol of cell viability assay. Only the

concentration 500 µg/ml presented a profile, as shown in Fig. 5. Moreover, when we

evaluated inflammatory mediators, CP (25, 50, and 100 mg/kg) also significantly

decreased the levels of TNF-α (p < 0.001) and IL-1β (p < 0.01 and p < 0.05) in the

pleural exudates collected at 4 h after carrageenan injection (Fig. 6A, B). The same

occurred with vascular leakage, once the CP, at doses of 25 and 100 mg/kg,

significantly decreased (p < 0.01) the number of proteins in plasma (Fig. 6C).

3.9 Effect of CP on Measurement of paw edema

As shown in Fig. 7A, CG injection increased mice paw volumes. Additionally,

treatment with CP significantly (p < 0.05, p < 0.01 and p < 0.001) decreased the edema.

At 50 and 100 mg/kg, CP, as well as indomethacin (10 mg/kg), was able to maintain

reduction of the edema during the six-hour evaluation period. CP percentages of

inhibition, based on the AUC values, were 26.4%, 49.8% (p < 0.01), and 56.6% (p <

0.001) for 25, 50, and 100 mg/kg, respectively, while indomethacin showed an

inhibition of 55.3% (p < 0.001) (Fig. 7B).

69

3.10 Effect of CP on spontaneous locomotor activity and Rota Rod

The effect of CP in the animal coordination was tested through the spontaneous

locomotor activity and the rota rod. In either test, it has been proved that the CP does

not alter the coordination of the animals, unlike DZP, which altered the ambulation

(number of crossings) and the ability to stay on the rota rod in the times of 0.5, 1, and 2

h after the treatment (data not shown).

4.0 Discussion

This study aims at evaluating the analgesic and anti-inflammatory effects of a

synthetic drug, obtained through an esterification reaction of the monoterpene carvacrol.

In recent years, studies have showed that carvacrol has anti-inflammatory effect

probably due to the inhibition of mediators such as PGE2, IL-1β and TNF-α [21, 31].

However, in these studies, carvacrol at lower doses seemed to be ineffective. Thus, the

structural modification in carvacrol could improve the action of this monoterpene.

Chemical modification of carvacrol monoterpenoids to ester derivatives has

already been performed to evaluate the antimicrobial and antifungal activity [19, 32].

However, the antinociceptive, hyperalgesic and anti-inflammatory activities of CP have

not yet been studied. Therefore, carvacrol was used as a starting material for the

synthesis of CP according to the literature with some modifications [22]. Formation of

CP was confirmed by the ultraviolet spectrum, mass spectra and nuclear magnetic

resonance as previously reported by Mathela et al. [19].

As no literature data regarding CP antinociceptive activity were found, the first

experimental protocol conducted to evaluate the effect of CP was nociception tests

induced by formalin and hot plate, in mice, protocols widely used in the literature. The

test of formalin-induced nociception involves a continuous and moderate pain from the

70

injured tissue, such feature distinguishes it from other existing tests of nociception [33].

Two phases are present in the test, namely the initial phase, which seems to be related to

direct activation by neurogenic stimulation of C fibers, mediated by substance P, and

late phase, which depends on the activation of nociceptive afferent neurons as well as

the release of Prostaglandin E2, nitric oxide (NO), tachykinins, kinins and other

inflammatory mediators [23, 34].

Previous studies prove that the formalin is an important agonist of channels in

this family of receptors, transient-receptor-potential subfamily 1 (TRPA1) [35], besides

the involvement of glutamatergic receptors AMPA and NMDA receptors in the acute

phase in the late phase of the test nociception induced by formalin. The activation of

these receptors linked to glutamatergic pathway implies a probable interaction with the

nociceptive pathway [36]. Such information suggests as a possible mechanism for the

antinociceptive action of CP acting on the TRPA1 receptors, NMDA and AMPA

receptors, since this compound had a significant effect in both phases of the test.

After application of a thermal stimulus, Aβ nerve fibers are activated and the

information is carried to the brain. When this same thermal stimulus presents an

noxious aspect shall, it activates the nerve fibers Aδ and C and the information is

carried to the brain [1], as occurs in the hot-plate test, which makes it suitable for the

screening of substances with analgesic activity center [37].

The VR1 receptor, present on nerve fiber Aδ and C may be activated when the

thermal exposure is at approximately 43°C and which, consequently, leads to the

opening of calcium channels [38, 39]. One possible explanation for the increase in the

time response in the hot-plate test in the animals treated with CP is the activation of this

receptor. Carvacrol, only at the highest dose (100mg/kg), showed a central analgesic

effect [40]. Thus, the obtained result indicates that the modification in the structure of

71

carvacrol contributed to the analgesic activity, since the CP had an effect at doses of 50

and 100 mg/kg. However, molecular studies could further elucidate this mechanism.

Hyperalgesia induced by injection of carrageenan, in animal models, is widely

used for evaluating new antihyperalgesic drugs in rodents. The CG, in animal models,

stimulates various cell types, particularly the resident and migratory cells to produce a

cascade of cytokines [41]. The first cytokine released is the TNF-α, which triggers the

release of IL-1β and keratinocyte-derived chemokine (KC) responsible for the synthesis

stimulation of prostaglandins and the release of the sympathetic amines, respectively

[42]. These final mediators will act on the nerve endings, but specifically on

metabotropic receptors to trigger the activation of second messenger pathways leading

to a decrease in cellular excitability threshold [43]. In this state, nociceptor activation

and impulse transmission by the primary nociceptive neurons are facilitated; in response

to that, the animals withdraw the paw with a force which is lower than the baseline

threshold.

In this protocol, the CP, at all doses, increased the animal sensitivity threshold,

as it happened with indomethacin, a cyclooxygenase inhibitor. Such effect can be

related to a possible inhibition of cytokine cascade. This inhibition may occur at the

level of the enzyme cyclooxygenase. Similarly, carvacrol inhibits the enzyme

cyclooxygenase-2 [44] and in larger doses (50 and 100 mg/kg) also has anti-

hyperalgesic effect [21, 40].

The TNF-α is further associated with the development of inflammatory pain

since it interacts with target cells through high-affinity membrane receptors, such as

TNF receptor Type 1 (TNFR1 or p55) and Type 2 (TNFR2 or p75) [45], stimulating the

secretion of IL-1β [46] and consequently, inducing the expression of COX-2,

responsible for various prostanoid biosynthesis, as PGE2 [47].

72

It was shown that the hyperalgesia induced by the injection of TNF-α was

reduced with administration of CP, at all doses. This reduction was also demonstrated

with indomethacin. Such results corroborate the idea of a possible COX-level inhibition,

without, however, ruling out a possible interaction at the level of receptor. Especially

with the receptor TNFR1, according to Sommer et al. [48] and Verri et al. [45] TNF-α

interacts with TNFR1 and triggers the hyperalgesic cascade.

Nevertheless, another hypothesis that was verified to evaluate the possible

mechanism of action of CP involves the blockade of sensitization or activation of the

nociceptor through the evaluation of its effect on hyperalgesia induced by PGE2 and

DA. These inflammatory mediators induce hyperalgesia by activating mainly receptors

present in nociceptor membranes, EP2 and D1, respectively, triggering their

sensitization [49]. By increasing the concentration of cAMP as well as the PKA

signaling pathways and/or PKC [50], which in turn catalyze phosphorylation reactions

resistant to sodium channels [51], phosphorylation of these channels changes the

conductance that increases neuronal excitability, thereby contributing to the induction of

inflammatory hyperalgesia [52].

As CP inhibited the hyperalgesia induced by PGE2 and DA, we are led to believe

that there is a possible involvement with the receptors present on neuronal membranes

(EP2 and D1). This action may even relate to structural modifications made to the

structure of carvacrol, since carvacrol was not able to inhibit hyperalgesia induced by

these agents [21]. The inhibition of hyperalgesia was also seen with the positive control,

dipyrone. One of the proposed mechanisms for dipyrone is in the activation of arginine-

NO-cGMP-channel ATP-sensitive K+, which induces desensitization of peripheral

nociceptors [53].

73

According to Cunha et al. [54], during the inflammatory process, neutrophils

actively participate in the hyperalgesic cascade activation with the induction training of

final mediators. Effects of hyperalgesic cytokines depend on neutrophil migration and

the ability of these cells to release direct-acting mediators such as PGE2.

Therefore, the blockade of neutrophil migration could be a target for the

development of new drugs, not only anti-inflammatory but analgesic as well. For this

reason, and to better investigate the anti-inflammatory and anti-hyperalgesic potential of

CP, we performed a cell migration test through carrageenan-induced pleurisy. The

results allowed us to detect a marked inhibitory effect of CP on neutrophil and

mononuclear cell migration, without altering the morphological profile of these cells,

what rules out the possibility of cytotoxicity.

CP has anti-inflammatory and anti-hyperalgesic properties since it reduces

neuronal excitability threshold and also inhibits the migration of neutrophils, thereby

reducing the inflammatory pain. Since the participation of neutrophils in this process

has been extensively studied, the pronociceptive action of neutrophils was first

suggested almost 35 years ago [55] and since then, several studies have reported the

importance of neutrophils in the pathogenesis of inflammatory pain [56-59].

Considering that cytokines, TNF-α and IL-1β, play key roles in inflammatory

processes, they stimulate the recruitment of neutrophils and monocytes to the sites of

infection and activate these cells to eradicate microorganisms [60]. Although there is

evidence to support a direct action of these cytokines on nociceptors, their primary

contribution to pain hypersensitivity results from potentiation of the inflammatory

response and increased production of algesic agents such as prostaglandins, bradykinin,

and extracellular protons [1].

74

In this way, there is the need to quantify these cytokines after an inflammatory

process induced by carrageenan. Corroborating previous results, CP decreased the levels

of TNF and IL-1, what leads us to believe that CP has a satisfactory anti-inflammatory

effect, since these cytokines are important in severe inflammatory conditions.

In addition to the characteristics of the inflammatory processes mentioned

above, such as cell migration, cytokine release has also extravasation of plasma fluid,

rich in proteins. This parameter was also evaluated and CP was effective against plasma

extravasation in dose of 25 and 100 mg/kg. Therefore, CP has satisfactory anti-

inflammatory effect, since it has decreased the essential factors in inflammatory

process.

The model of paw edema induced by carrageenan is widely used by the

scientific community with the goal of potential drug discovery with anti-inflammatory

activity. The carrageenan induces a biphasic response. In the first hours after

administration of the agent, the edema is mediated by the early release of histamine and

serotonin followed by the release of kinin and finally through the release of bradykinin

and prostaglandins (PGs) [61, 62]. According to the result of our study, CP, in doses of

50 and 100 mg/kg, was able to effectively inhibit the edema throughout the observation

period, suggesting that CP inhibits different chemical mediators of inflammation.

Interestingly, the anti-inflammatory nature of CP was similar with to carvacrol

as described by Guimarães et al. [21], which leads us to believe that the addition of a

propionyl group does not alter the anti-inflammatory activity of carvacrol. However, in

respect to its action on neural stimulation, as demonstrated in protocols in hyperalgesia

induced by PGE2 and DA, the addition in this group had to be limited since carvacrol

had no positive effect positive in this protocol, according to Guimarães et al. [21].

75

As shown by Passos et al. [63], many terpenoids have activity on the central

nervous system (CNS) due to the inhibitory effect on the CNS or muscle relaxation.

Thus, these activities could reduce the motor coordination of animals and invalidate the

results obtained for the CP. Therefore, it was necessary to evaluate the effect of CP on

the CNS.

As shown, the CP did not alter the spontaneous movement and coordination of

animals, which leads one to believe that the CP has no inhibitory effect on the CNS.

Since mobility is a function of the degree of excitability of the central nervous system

and a decrease of this parameter is suggestive of a depressive activity [64], the animals

have remained on the rotating bar during the time set, discarding thus the possibility of

a myorelaxing effect [65].

Thus, it can be concluded that CP is effective as an analgesic and anti-

inflammatory compound in various pain models, probably mediated via inhibition of

peripheral mediators (as TNF-α and IL-1β synthesis) as well as central inhibitory

mechanisms. Nevertheless, further studies are necessary to understand the precise

mechanisms of action of CP on inflammatory pain.

Acknowledgments

This work was supported by grants from Conselho Nacional de Desenvolvimento

Científico e Tecnológico (CNPq/Brazil), Funda o de Apoio Pesquisa e Inova o

Tecnol gica do stado de Sergipe (FAPIT C/S / razil) and Financiadora de Estudos e

Projetos (FINEP/Brazil). We thank teacher Abilio Borghi for the grammar review on

the manuscript.

Declaration of interest: The authors report no conflicts of interest.

76

References

1. Basbaum, A.I., D.M. Bautista, G. Scherrer, and D. Julius. 2009. Cellular and

molecular mechanisms of pain. Cell 139(2): 267-284.

2. McMahon, S.B., D.L.H. Bennett, and S. Bevan. 2008. Inflammatory mediators and

modulators of pain. In: McMahon, S.B., M. Koltzenburg, Ed. Wall and Melzack's

textbook of Pain. Elsevier: pp. 49-72.

3. Rao, P., and E.E. Knaus. 2008. Evolution of nonsteroidal anti-inflammatory drugs

(NSAIDs): cyclooxygenase (COX) inhibition and beyond. Journal of Pharmaceutical

Sciences 11(2): 81-110.

4. Cunha, T.M., D. Roman-Campos, C.M. Lotufo, H.L. Duarte, G.R. Souza, W.A. Verri

Jr, M.I. Funez, Q.M. Dias, I.R. Schivo, A.C. Domingues, D. Sachs, S. Chiavegatto,

M.M. Teixeira, J.S. Hothersall, J.S. Cruz, F.Q. Cunha, and S.H. Ferreira. 2010.

Morphine peripheral analgesia depends on activation of the

PI3Kgamma/AKT/nNOS/NO/KATP signaling pathway. Proceedings of the National

Academy of Sciences USA 107(9): 4442-4447.

5. Furlan, A.D., J.A. Sandoval, A. Mailis-Gagnon, and E. Tunks. 2006. Opioids for

chronic noncancer pain: a meta-analysis of effectiveness and side effects. Canadian

Medical Association Journal 174(11): 1589-1594.

6. Vane, J.R., Y.S. Bakhle, and R.M. Botting. 1998. Cyclooxygenases 1 and 2. Annu

Annual Review of Pharmacology and Toxicology 38: 97-120.

7. De Cassia da Silveira e Sa, R., L.N. Andrade, and D.P. De Sousa. 2013. A review on

anti-inflammatory activity of monoterpenes. Molecules 18(1):1227-1254.

8. Guimarães, A.G., J.S.S. Quintans, and L.J. Quintans-Júnior. 2013. Monoterpenes

with analgesic activity-a systematic review. Phytotherapy Research 27(1): 1-15.

9. Baker, D.D., M. Chu, U. Oza, and V. Rajgarhia. 2007. The value of natural products

to future pharmaceutical discovery. Natural Product Reports 24(6): 1225-1244.

10. Ganesan, A. 2008. The impact of natural products upon modern drug discovery.

Current Opinion in Chemical Biology 12(3): 306-317.

11. Newman, D.J., and G.M. Cragg. 2012. Natural products as sources of new drugs

over the 30 years from 1981 to 2010. Journal of Natural Products 75(3): 311-335.

12. Harvey, A.L. 2008. Natural products in drug discovery. Drug Discovery Today

13(19-20): 894-901.

13. Porto, T.S., N.A. Furtado, V.C. Heleno, C.H. Martins, F.B. Da Costa, M.E.

Severiano, A.N. Silva, R.C. Veneziani, and S.R. Ambrósio. 2009. Antimicrobial ent-

pimarane diterpenes from Viguiera arenaria against Gram-positive bacteria. Fitoterapia

80(7): 432-436.

14. Olagnier, D., P. Costes, A. Berry, M.D. Linas, M. Urrutigoity, O. Dechy-Cabaret,

and F. Benoit-Vical. 2007. Modifications of the chemical structure of terpenes in

antiplasmodial and antifungal drug research. Bioorganic & Medicinal Chemistry Letters

17(22): 6075-6078.

15. De Sousa, D.P., E.A. Camargo, F.S. Oliveira, and R.N. De Almeida. 2010. Anti-

inflammatory activity of hydroxydihydrocarvone. Zeitschrift für Naturforschung C

65(9-10): 543-550.

16. Oliveira, F.S., D.P. De Sousa, and R.N. De Almeida. 2008. Antinociceptive effect of

hydroxydihydrocarvone. Biological & Pharmaceutical Bulletin 31(4): 588-591.

17. Goncalves, J.C., A.M. Alves, A.E. De Araujo, J.S. Cruz, and D.A. Araujo. 2010.

Distinct effects of carvone analogues on the isolated nerve of rats. European Journal of

Pharmacology 645(1-3): 108-112.

77

18. De Sousa, D.P., E.V. Junior, F.S. Oliveira, R.N. De Almeida, X.P. Nunes, and J.M.

Barbosa-Filho. 2007. Antinociceptive activity of structural analogues of rotundifolone:

structure-activity relationship. Zeitschrift für Naturforschung C 62(1-2): 39-42.

19. Mathela, C.S., K.K. Singh, and V.K. Gupta. 2010. Synthesis and in vitro

antibacterial activity of thymol and carvacrol derivatives. Acta Poloniae Pharmaceutica

67(4): 375-380.

20. Quintans-Júnior, L.J., M.S. Melo, D.P. De Sousa, A.A. Araujo, A.C. Onofre, D.P.

Gelain, J.C. Gonçalves, D.A. Araújo, J.R. Almeida, and L.R. Bonjardim. 2010.

Antinociceptive effects of citronellal in formalin-, capsaicin-, and glutamate-induced

orofacial nociception in rodents and its action on nerve excitability. Journal of

Orofacial Pain 24(3): 305-312.

21. Guimarães, A.G., M.A. Xavier, M.T. De Santana, E.A. Camargo, C.A. Santos, F.A.

Brito, E.O. Barreto, S.C. Cavalcanti, A.R. Antoniolli, R.C. Oliveira, and L.J. Quintans-

Júnior. 2012. Carvacrol attenuates mechanical hypernociception and inflammatory

response. Naunyn-Schmiedeberg's Archives of Pharmacology 385(3): 253-263.

22. Dolly, B.B., and F. Barba. 2003. Cathodic reduction of hydroxycarbonyl compound

trichloroacetyl esters. Tetrahedron 59: 9161-9165.

23. Hunskaar, S., and K. Hole. 1987. The formalin test in mice: dissociation between

inflammatory and non-inflammatory pain. Pain 30(1): 103-114.

24. Kuraishi, Y., Y. Harada, S. Aratani, M. Satoh, and H. Takagi. 1983. Separate

involvement of the spinal noradrenergic and serotonergic systems in morphine

analgesia: the differences in mechanical and thermal algesic tests. Brain Research

273(2): 245-252.

25. Cunha, T.M., W.A. Verri Jr, G.G. Vivancos, I.F. Moreira, S. Reis, C.A. Parada, F.Q.

Cunha, and S.H. Ferreira. 2004. An electronic pressure-meter nociception paw test for

mice. Brazilian Journal of Medical and Biological Research 37(3): 401-407.

26. Villarreal, C.F., M.I. Funez, F. Figueiredo, F.Q. Cunha, C.A. Parada, and S.H.

Ferreira. 2009. Acute and persistent nociceptive paw sensitisation in mice: the

involvement of distinct signalling pathways. Life Sciences 85(23-26): 822-829.

27. Mosmann, T. 1983. Rapid colorimetric assay for cellular growth and survival:

application to proliferation and cytotoxicity assays. Journal of Immunological Methods

65(1-2): 55-63.

28. Levy, L. 1969. Carrageenan paw edema in the mouse. Life Sciences 8(11): 601-606.

29. Asakura, W., K. Matsumoto, H. Ohta, and H. Watanabe. 1993. Effect of alpha 2-

adrenergic drugs on REM sleep deprivation-induced increase in swimming activity.

Pharmacology Biochemistry and Behavior 46(1): 111-115.

30. Dunham, N.W., and T.S. Miya. 1957. A note on a simple apparatus for detecting

neurological deficit in rats and mice. Journal of American Pharmacists Association

46(3): 208-209.

31. Lima Mda, S., L.J. Quintans-Junior, W.A. De Santana, C. Martins Kaneto, M.B.

Pereira Soares, and C.F. Villarreal. 2013. Anti-inflammatory effects of carvacrol:

evidence for a key role of interleukin-10. European Journal of Pharmacology 699(1-3):

112-117.

32. Nikumbh, V.P., V.S. Tare, and P.P. Mahulikar. 2003. Ecofriendly pest management

using monoterpenoids-III: Antifungal efficacy of carvacrol derivatives. Journal of

Scientific & Industrial Research 62: 1086-1089.

33. Zouikr, I., M.A. Tadros, V.L. Clifton, K.W. Beagley, and D.M. Hodgson. 2013.

Low formalin concentrations induce fine-tuned responses that are sex and age-

dependent: a developmental study. PloS one 8(1): e53384.

78

34. Capuano, A., A. De Corato, M. Treglia, G. Tringali, C. Dello Russo, and P. Navarra.

2009. Antinociceptive activity of buprenorphine and lumiracoxib in the rat orofacial

formalin test: a combination analysis study. European Journal of Pharmacology 605(1-

3): 57-62.

35. Mogil, J.S. 2009. Animal models of pain: progress and challenges. Nature Reviews

Neuroscience 10(4): 283-294.

36. Hunter, J.C., and L. Singh. 1994. Role of excitatory amino acid receptors in the

mediation of the nociceptive response to formalin in the rat. Neuroscience Letters

174(2): 217-221.

37. Le Bars, D., M. Gozariu, and S.W. Cadden. 2001. Animal models of nociception.

Pharmacological Reviews 53(4): 597-652.

38. Davis, J.B., J. Gray, M.J. Gunthorpe, J.P. Hatcher, P.T. Davey, P. Overend, M.H.

Harries, J. Latcham, C. Clapham, K. Atkinson, S.A. Hughes, K. Rance, E. Grau, A.J.

Harper, P.L. Pugh, D.C. Rogers, S. Bingham, A. Randall, and S.A. Sheardown. 2000.

Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia. Nature

405(6783): 183-187.

39. Caterina, M.J., M.A. Schumacher, M. Tominaga, T.A. Rosen, J.D. Levine, and D.

Julius. 1997. The capsaicin receptor: a heat-activated ion channel in the pain pathway.

Nature 389(6653): 816-824.

40. Guimarães, A.G., G.F. Oliveira, M.S. Melo, S.C. Cavalcanti, A.R. Antoniolli, L.R.

Bonjardim, F.A. Silva, J.P. Santos, R.F. Rocha, J.C. Moreira, A.A. Araújo, D.P. Gelain,

and L.J. Quintans-Júnior. 2010. Bioassay-guided evaluation of antioxidant and

antinociceptive activities of carvacrol. Basic & Clinical Pharmacology & Toxicology

107(6): 949-957.

41. Ferreira, S.H., B.B. Lorenzetti, and S. Poole. 1993. Bradykinin initiates cytokine-

mediated inflammatory hyperalgesia. British journal of pharmacology 110(3):1227-

1231.

42. Cunha, F.Q., B.B. Lorenzetti, S. Poole, and S.H. Ferreira. 1991. Interleukin-8 as a

mediator of sympathetic pain. British journal of pharmacology 104(3): 765-767.

43. Petho, G., A. Derow, and P.W. Reeh. 2001. Bradykinin-induced nociceptor

sensitization to heat is mediated by cyclooxygenase products in isolated rat skin.

European Journal of Neuroscience 14(2): 210-218.

44. Landa, P., L. Kokoska, M. Pribylova, T. Vanek, and P. Marsik. 2009. In vitro anti-

inflammatory activity of carvacrol: Inhibitory effect on COX-2 catalyzed prostaglandin

E(2) biosynthesis. Archives of Pharmacal Research 32(1):75-78.

45. Verri Jr, W.A., T.M. Cunha, C.A. Parada, S. Poole, F.Q. Cunha, and S.H. Ferreira.

2006. Hypernociceptive role of cytokines and chemokines: targets for analgesic drug

development? Pharmacology & Therapeutics 112(1): 116-138.

46. Cunha, T.M., W.A. Verri Jr, D.A. Valerio, A.T. Guerrero, L.G. Nogueira, S.M.

Vieira, D.G. Souza, M.M. Teixeira, S. Poole, S.H. Ferreira, and F.Q. Cunha. 2008. Role

of cytokines in mediating mechanical hypernociception in a model of delayed-type

hypersensitivity in mice. European Journal of Pain 12(8): 1059-1068.

47. Cunha, T.M., W.A. Verri Jr, J.S. Silva, S. Poole, F.Q. Cunha, and S.H. Ferreira.

2005. A cascade of cytokines mediates mechanical inflammatory hypernociception in

mice. Proceedings of the National Academy of Sciences USA 102(5): 1755-1760.

48. Sommer, C., C. Schmidt, and A. George. 1998. Hyperalgesia in experimental

neuropathy is dependent on the TNF receptor 1. Experimental Neurology 151(1): 138-

142.

79

49. Cunha, F.Q., S. Poole, B.B. Lorenzetti, and S.H. Ferreira. 1992. The pivotal role of

tumour necrosis factor alpha in the development of inflammatory hyperalgesia. British

Journal of Pharmacology 107(3): 660-664.

50. Chen, H.S., J. Lei, X. He, F. Qu, Y. Wang, W.W. Wen, H.J. You, and L. Arendt-

Nielsen. 2008. Peripheral involvement of PKA and PKC in subcutaneous bee venom-

induced persistent nociception, mechanical hyperalgesia, and inflammation in rats. Pain

135(1-2): 31-36.

51. Gold, M.S., D.B. Reichling, M.J. Shuster, and J.D. Levine. 1996. Hyperalgesic

agents increase a tetrodotoxin-resistant Na+ current in nociceptors. Proceedings of the

National Academy of Sciences USA 93(3): 1108-1112.

52. Lai, J., M.S. Gold, C.S. Kim, D. Bian, M.H. Ossipov, J.C. Hunter, and F. Porreca.

2002. Inhibition of neuropathic pain by decreased expression of the tetrodotoxin-

resistant sodium channel, NaV1.8. Pain 95(1-2): 143-152.

53. Lorenzetti, B.B., and S.H. Ferreira. 1996. Activation of the arginine-nitric oxide

pathway in primary sensory neurons contributes to dipyrone-induced spinal and

peripheral analgesia. Inflammation Research 45(6): 308-311.

54. Cunha, T.M., W.A. Verri Jr, I.R. Schivo, M.H. Napimoga, C.A. Parada, S. Poole,

M.M. Teixeira, S.H. Ferreira, and F.Q. Cunha. 2008. Crucial role of neutrophils in the

development of mechanical inflammatory hypernociception. Journal of Leukocyte

Biology 83(4): 824-832.

55. Castro, M.S.A., and S.H. Ferreira. 1979. Cell migration and hyperalgesia: a

paradoxical effect of endotoxin. in: Weissmann, G., B. Samuelsson, R. Paoletti, Eds.

Advances in Inflammation Research. Raven. New York: pp. 311-316.

56. Levine, J.D., W. Lau, G. Kwiat, and E.J. Goetzl. 1984. Leukotriene B4 produces

hyperalgesia that is dependent on polymorphonuclear leukocytes. Science 225(4663):

743-745.

57. Bennett, G., S. al-Rashed, J.R. Hoult, and S.D. Brain. 1998. Nerve growth factor

induced hyperalgesia in the rat hind paw is dependent on circulating neutrophils. Pain

77(3): 315-322.

58. Foster, P.A., S.K. Costa, R. Poston, J.R. Hoult, and S.D. Brain. 2003. Endothelial

cells play an essential role in the thermal hyperalgesia induced by nerve growth factor.

The FASEB Journal 17(12): 1703-1705.

59. Lavich, T.R., R. A. Siqueira, F.A. Farias-Filho, R.S. Cordeiro, P.M. Rodrigues e

Silva, and M.A. Martins. 2006. Neutrophil infiltration is implicated in the sustained

thermal hyperalgesic response evoked by allergen provocation in actively sensitized

rats. Pain 125(1-2): 180-187.

60. Nair, V., S. Singh, and Y.K. Gupta. 2012. Evaluation of disease modifying activity

of Coriandrum sativum in experimental models. Indian Journal of Medical Research

135: 240-245.

61. Posadas, I., M. Bucci, F. Roviezzo, A. Rossi, L. Parente, L. Sautebin, and G. Cirino.

2004. Carrageenan-induced mouse paw oedema is biphasic, age-weight dependent and

displays differential nitric oxide cyclooxygenase-2 expression. British Journal of

Pharmacology 142(2): 331-338.

62. Castro, J.A., H.A. Sasame, H. Sussman, and J.R. Gillette. 1968. Diverse effects of

SKF 525-A and antioxidants on carbon tetrachloride-induced changes in liver

microsomal P-450 content and ethylmorphine metabolism. Life Sciences 7(3): 129-136.

63. Passos, C.S., M.D. Arbo, S.M.K. Rates, and G.L. Von Poser. 2009.Terpenoids with

activity in the Central Nervous System (CNS). Brazilian Journal of Pharmacognosy

19(1): 140-149.

80

64. Franco, C.I., L.C. Morais, L.J. Quintans-Júnior, R.N. Almeida, and A.R. Antoniolli.

2005. CNS pharmacological effects of the hydroalcoholic extract of Sida cordifolia L.

leaves. Journal of Ethnopharmacology 98(3): 275-279.

65. Capasso, A., V. De Feo, F. De Simone, and L. Sorrentino. 1996. Pharmacology

effects of aqueous extract from Valeriana adscendens. Phytotherapy Research 10(4):

309-312.

81

FIGURES

1h, room temperature, anhydrous

Trirthylamine, CH3CH2COCl

O

O

OH

12

3

45

6

7

8

9 10

Figure 1. Sythesis reaction of the carvacrol propionate (CP) from the reagents

carvacrol, triethylamine and propionile chloride.

Vehicle 25 50 100 30

50

100

150

CP (mg/kg)

***

***

***

**

MOR

A

Lin

ckin

g t

ime (

s)

Veículo 25 50 100 30

50

100

150

***

******

***

B

CP (mg/kg) MOR

Lin

ckin

g t

ime (

s)

Figure 2. Effects of carvacrol proprionate (CP; 25, 50 or 100 mg/kg, i.p.) or morphine

(MOR, 3 mg/kg; i.p.) on formalin-induced nociceptive behavior were administered

intraperitoneally 0.5 hr before formalina injection. (panel A) First phase (0-5 min.) and

(panel B) second phase (15-30 min.) of the formalin test. Values represent mean ±

S.E.M. (n = 6, per group). **p < 0.01 and ***p < 0.001 versus control (one-way

ANOVA followed by Tukey’stest).

82

0 30 60 90 120 150 1800

4

8

12Vehicle

CP (25 mg/kg)

CP (50 mg/kg)

CP (100 mg/kg)

IND (10 mg/kg)

*** *** *** ***

***

****** ***

***

*** ***

***

***

***

***

*

Time (min)

Inte

nsit

y o

f h

yp

era

lgesia

(

of

wit

hd

raw

al

thre

sh

ol,

g)

0 30 60 90 120 150 1800

2

4

6

8Vehicle

CP (25 mg/kg)

CP (50 mg/kg)

CP (100 mg/kg)

IND (10 mg/kg)

******

***

***

**

***

**

****** ***

******

**

***

*

***

Time (min)

Inte

nsit

y o

f h

yp

ern

ocic

ep

tio

n

(

of

wit

hd

raw

al

thre

sh

old

, g

)

0

2

4

6

8

10Vehicle

CP (25 mg/kg)

CP (50 mg/kg)

CP (100 mg/kg)

DIP (60 mg/kg)

I I I I I

0 30 60 120 180

Time (min)

***

******

***

***

***

******

***

***

******

***

***

***

***

Inte

nsit

y o

f h

yp

ern

ocic

ep

tio

n

(

of

wit

hd

raw

al

thre

sh

old

, g

)

0

2

4

6

8

10Vehicle

CP (25 mg/kg)

CP (50 mg/kg)

CP (100 mg/kg)

DIP (60 mg/kg)

I I I I I

0 30 60 120 180

Time (min)

*** *** ******

*****

*** ***

***

********

*

Inte

nsit

y o

f h

yp

ern

ocic

ep

tio

n

(

of

wit

hd

raw

al

thre

sh

old

, g

)

A B

C D

Figure 3. Effect of acute administration of vehicle, carvacrol propionate (CP; 25, 50 or

100 mg/kg, i.p.), indomethacin (IND, 10 mg/kg, i.p.) or dipyrone (DIP, 60 mg/kg, i.p.)

on mechanical hypernociception induced by carrageenan (A), TNF-α ( ), PG 2 (C) and

dopamine (D). Each point represents the mean ± S.E.M. of the paw withdrawal

threshold (in grams) to tactile stimulation of the left hind paw. * p < 0.05, **p < 0.01

and ***p < 0.001 vs. control group (two-way-ANOVA followed by Bonferroni).

83

Saline Vehicle 25 50 100 100

2

4

6

8

10

12

*** *** ******

Carrageenan (300 g/cavity)

_______________ ___CP (mg/kg) IND

AT

ota

is l

eu

ko

cyte

s

(x 1

06

cell

s/c

avit

y)

Saline Vehicle 25 50 100 100

2

4

6

8

10

12

Carrageenan (300 g/cavity)

CP (mg/kg) IND

_______________ ___

*** *** ***

*

B

Neu

tro

ph

ilis

(x 1

06

cell

s/c

avit

y)

Figure 4. Effect of acute administration of vehicle, carvacrol propionate (CP; 25, 50 or

100 mg/kg, i.p.) or indomethacin (IND, 10 mg/kg; i.p.) on the inflammation by

carrageenan in mice pleurisy. The analyses were performed 4 h after carrageenan

injection (300 μg/cavity) to evaluate the recruitment of total leukocytes (A), neutrophils

(B). Data were expressed as mean ± SEM, for a minimum of six animals. * p < 0.05, **

p < 0.01, and *** p < 0.001 compared with the control group (vehicle) (ANOVA

followed by Tukey test).

3 1 10 100 250 5000

50

100

CP (g/ml)

**

Tween (%)

_____ ____________________________

Cell V

iabilit

y (

%)

Figure 5. Effect of vehicle, carvacrol propionate (CP; 1, 10, 100, 250 or 500 µg/mL, in

vitro) on murine peritoneal macrophages (2.5×105 cells). The percentage of viability

was determined in relation to controls. Data were expressed as mean ± SEM. ** p <

0.01 compared with the control group (vehicle) (ANOVA followed by Tukey test).

84

Saline Vehicle 25 50 100 100

100

200

300

400

500

600

*** *** ******

Carrageenan (300 g/cavity)

CP (mg/kg) IND

_______________ ___

AT

NF

- (

pg

/ml)

Saline Vehicle 25 50 100 100

200

400

600

*****

**

Carrageenan (300 g/cavity)

CP (mg/kg) IND

_______________ ___

B

IL-

1b

eta

(p

g/m

l)

Saline Vehicle 25 50 100 100

4

8

12

16

** ** **

Carrageenan (300 g/cavity)

CP (mg/kg) IND

_______________ ___

C

To

tal

pro

tein

(

g/m

l)

Figure 6. Effect of acute administration of vehicle, carvacrol propionate (CP; 25, 50 or

100 mg/kg, i.p.) or indomethacin (IND, 10 mg/kg; i.p.) on the inflammation by

carrageenan in mice pleurisy. The analyses were performed 4 h after carrageenan injection

(300 μg/cavity) to evaluate to assess tumor necrosis factor-alpha (TNF-α) (A), and

interleukin-1β (IL-1β) levels (B), and total protein (C). Data were expressed as mean ±

SEM, for a minimum of six animals. * p < 0.05, ** p < 0.01, and *** p <0.001 compared

with the control group (vehicle) (ANOVA followed by Tukey test).

85

0 1 2 3 4 5 60.00

0.05

0.10

0.15

0.20 Vehicle

CP (25 mg/kg)

CP (50 mg/kg)

CP (100 mg/kg)

IND (10 mg/kg)

**

***

***

***

* ******

*** ***

***

***

*********

**

*

**

*

Time (h)

Ed

em

a (

mL

)

Vehicle 25 50 100 100.0

0.2

0.4

0.6

0.8

1.0

CP (mg/kg)

***** ***

_____________

IND

AU

C (

0-6

h)

A B

Figure 7. Effect of acute administration of vehicle, carvacrol proprionate (CP; 25, 50 or

100 mg/kg, i.p.) or indomethacin (IND, 10 mg/kg; i.p.) on edema induced by carrageenan.

Each point represents the mean±SEM of the paw volume (in milliliter, panel A) or the area

under curve (AUC) from 0 to 6 h (panel B). *p < 0.05, **p < 0.01 and ***p < 0.001 vs.

control group (two-way-ANOVA followed by Bonferroni – panel A and ANOVA

followed by Tukey test – panel B).

86

Table 1. Effect of CP (25, 50, or 100 mg/kg; i.p.) or MOR (3.0 mg/kg; i.p.) on the hot

plate test in mice.

Treatment Dose

(mg/kg)

Reaction time (licking of the hind paws) (s)a

Basal 0.5h 1h 1.5h 2h

Vehicle - 7.0 ± 0.68 8.7 ± 0.42 7.7 ± 0.33 6.7 ± 0.21 5.7 ± 0.71

CP 25 7.3 ± 0.67 10.0 ± 0.58 10.8 ± 1.25 9.5 ± 0.96

10.0 ± 0.52

CP 50 8.0 ± 0.45 14.2 ± 1.3d 16.5 ± 2.3

c 15.7 ± 2.0

d 16.7 ± 1.8

d

CP 100 7.8 ± 0.70 12.2 ± 0.5b 13.8 ± 1.3

b 12.3 ± 1.1

b 15.5 ± 1.5

d

MOR 3 7.6 ± 1.9 30.0 ± 0.0 d 29.5 ± 0.4

d 29.0 ± 0.9

d 22.7 ± 4.9

d

Values are the mean ± SEM (n = 6, per group)

a Values represent mean S.E.M.

b p < 0.05 as compared with control (vehicle) (ANOVA followed by Tukey test).

c p < 0.01 as compared with control (vehicle) (ANOVA followed by Tukey test).

d p < 0.001 as compared with control (vehicle) (ANOVA followed by Tukey test).

87

4.0 CONCLUSÃO

88

4.0 CONSIDERAÇÕES FINAIS

Tendo em vista os resultados obtidos no presente estudo, pode-se concluir:

CAPÍTULO 1

Modificação estrutural em terpenos representa uma ferramenta

farmacológica para a descoberta de drogas com ação anti-inflamatória;

CAPÍTULO 2

O propionato de carvacrol foi sintetizado e identificado;

Apresenta ação antinociceptiva, sendo capaz de reduzir a nocicepção em

roedores;

Tem efeito anti-hiperalgésico, já que inibe a cascata hiperalgésica;

Possui efeito anti-inflamatório, provavelmente mediado pela inibição de

citocinas pró-inflamatórias, a exemplo do TNF-α e IL-1β;

Não apresenta citotoxicidade celular;

Nas doses utilizadas não induz qualquer alteração na coordenação motora

dos animais.

DISSERTAÇÃO

Os dados apresentados no presente estudo nos permitem sugerir que a

semi-síntese de monoterpenos pode ser útil para a descoberta de drogas

com possível ação anti-inflamatória.

Novas metodologias podem ser propostas para melhor caracterizar o

mecanismo exato do CP.

89

ANEXOS

90

Anexo 1: PROTOCOLO DE APROVAÇÃO NO COMITÊ DE ÉTICA EM

PESQUISA ANIMAL DA UNIVERSIDADE FEDERAL DE SERGIPE

91

Anexo 2: CERTIFICADO DE HONRA AO MÉRITO

92

Anexo 3: CERTIFICADO DE HONRA AO MÉRITO

93

Anexo 4: ACEITE DO PERIÓDICO INFLAMMATION

94

ANEXO 5: ESPECTROS DE RMN DO PROPIONATO DE CARVAROL Espectro de RMN de 1H do propionato de carvacrol (400 MHz, CDCl3)

ppm (f1)

0.01.73.35.06.78.3

7.1

17

9

7.0

98

3

6.9

93

3

6.9

88

9

6.9

73

8

6.9

69

4

6.8

58

0

6.8

53

7

2.9

01

0

2.8

83

7

2.8

66

4

2.8

49

1

2.8

31

9

2.8

14

6

2.7

97

4

2.5

93

3

2.5

74

3

2.5

55

4

2.5

36

6

2.1

09

9

1.2

75

2

1.2

56

3

1.2

37

4

1.2

23

7

1.2

06

4

0.0

00

0

1.0

01

.00

0.9

7

1.0

2

2.0

7

3.0

7

3.1

73

.16

3.1

0

95

ppm (f1)

6.8506.9006.9507.0007.0507.100

7.1

17

9

7.0

98

3

6.9

93

3

6.9

88

9

6.9

73

8

6.9

69

4

6.8

58

0

6.8

53

7

1.0

0

1.0

0

0.9

7

ppm (f1)

2.5502.6002.6502.7002.7502.8002.8502.900

2.9

01

0

2.8

83

7

2.8

66

4

2.8

49

1

2.8

31

9

2.8

14

6

2.7

97

4

2.5

93

3

2.5

74

3

2.5

55

4

2.5

36

6

1.0

2

2.0

7

96

ppm (f1)

1.201.301.401.501.601.701.801.902.002.10

2.1

09

9

1.2

75

2

1.2

56

3

1.2

37

4

1.2

23

7

1.2

06

4

3.0

7

3.1

7

3.1

6

3.1

0

ppm (f1)

1.2001.2101.2201.2301.2401.2501.2601.2701.280

1.2

75

2

1.2

56

3

1.2

37

4

1.2

23

7

1.2

06

4

3.1

7

3.1

6

3.1

0

97

Espectro de RMN de 13C do propionato de carvacrol (100 MHz, CDCl3)

ppm (f1)

0255075100125150175200225

17

2.5

8

14

9.3

7

14

7.9

8

13

0.8

5

12

7.1

5

12

4.0

0

11

9.7

9

77

.52

77

.20

76

.88

33

.60

27

.60

23

.92

15

.74

9.2

7

-0.0

00

00